Composite materials comprising supported porous gels

ABSTRACT

This invention relates to a composite material that comprises a support member that has a plurality of pores extending through the support member and, located in the pores of the support member, and filling the pores of the support member, a macroporous cross-linked gel. The invention also relates to a process for preparing the composite material described above, and to its use. The composite material is suitable, for example, for separation of substances, for example by filtration or adsorption, including chromatography, for use as a support in synthesis or for use as a support for cell growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patentapplication Serial No. 60/447,730, filed Feb. 19, 2003, entitled“COMPOSITE MATERIALS COMPRISING SUPPORTED POROUS GELS”, the contents ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to composite materials that comprisesupported macroporous cross-linked gels, and to their preparation anduse. The composite materials are suitable, for example, for separationof substances, for example by filtration or adsorption, includingchromatography, for use as a support in synthesis or for use as asupport for cell growth.

BACKGROUND OF THE INVENTION

[0003] Composite materials and separation materials have been describedin patent documents such as:

[0004] U.S. Pat. Nos. 4,224,415; 4,889,632; 4,923,610; 4,952,349;5,160,627; November 1992; U.S. Pat. Nos. 5,593,576; 5,599,453;5,672,276; 5,723,601; 5,906,734; 6,045,697; 6,086,769; and 6,258,276;

[0005] International Patent Nos. EP 316,642; WO 00/12618; WO 00/50160;EP 316,642 B1; and EP 664,732 B1;

[0006] and in other publications, for example:

[0007] Liu, H. C. and Fried, J. R., Breakthrough of lysozyme through anaffinity membrane of cellulose-Cibaron Blue. AIChE Journal, vol. 40(1994), p. 40-49.

[0008] Tennikov, M. B.; Gazdina, N. V.; Tennikova, T. B.; Svec, F.,Effect of porous structure of macroporous polymer supports on resolutionin high-performance membrane chromatography. Journal of ChromatographyA, vol. 798 (1998) p. 55-64.

[0009] Svec, F.; Jelinkova, M.; Votavova, E., Reactive macroporousmembranes based on glycidyl methacrylate-ethylene dimethacrylatecopolymer for high-performance membrane chromatography. Angew. Makromol.Chem. Vol. 188 (1991) p. 167-176.

[0010] Tennikova, T. B.; Belenkii, B. G.; Svec, F., High-performancemembrane chromatography. A novel method of protein separation. J. LiquidChromatography, vol. 13 (1990) p. 63-70.

[0011] Tennikova, T. B.; Bleha,M.; Svec, F.; Almazova, T. V.; Belenkii,B. G. J., High-performance membrane chromatography of proteins, a novelmethod of protein separation. Chromatography, vol. 555 (1991) p. 97-107.

[0012] Tennikova, T. B.; Svec, F. High-performance membranechromatography: highly efficient separation method for proteins inion-exchange, hydrophobic interaction and reversed-phase modes. J.Chromatography, vol. 646 (1993) p. 279-288.

[0013] Viklund, C.; Svec, F.; Fréchet, J. M. J. Fast ion-exchange HPLCof proteins using porous poly(glycidyl methacrylate-co-ethylenedimethacrylate) monoliths grafted withpoly(2-acrylamido-2-methyl-1-propanesulfonic acid). Biotechnol.Progress, vol. 13 (1997) p. 597-600.

[0014] Mika, A. M. and Childs, R. F. Calculation of the hydrodynamicpermeability of gels and gel-filled microporous membranes, Ind. Eng.Chem. Res., vol. 40 (2001), p. 1694-1705.

BRIEF SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides a compositematerial that comprises a support member that has a plurality of poresextending through the support member and, located in the pores of thesupport member and essentially filling the pores of the support member,a macroporous cross-linked gel. In some embodiments, the macroporous gelused is responsive to environmental conditions, providing a responsivecomposite material.

[0016] In another aspect, the invention provides a process for theseparation of substances by means of the composite material describedabove.

[0017] In another aspect, the invention provides a process for solidphase chemical synthesis, wherein the composite material serves as thesolid phase in the pores of which the chemical synthesis occurs.

[0018] In another aspect, the invention provides a process for growth ofa microorganism or cell, wherein the composite material serves as asolid support in the pores of which the growth occurs.

[0019] In yet another aspect, the invention provides a process forpreparing the composite material described above, the processcomprising:

[0020] a) introducing into the pores of the support member a solution orsuspension containing

[0021] i) one or more monomers and one or more cross-linking agents thatcan combine to form a macroporous gel, or

[0022] ii) one or more cross-linkable polymers and one or morecross-linking agents that can combine to form a macroporous gel,

[0023] b) reacting the monomers and the cross-linking agents or thepolymers and the cross-linking agents to form a macroporous cross-linkedgel that fills the pores of the support member.

[0024] The macroporous gel fills the pores of the support laterally,i.e. substantially perpendicular to the direction of the flow throughthe composite material. By “fill” we mean that, in use, essentially allliquid that passes through the composite material must pass through themacroporous gel. A support member whose pores contain macroporous gel tosuch an amount that this condition is satisfied is regarded as filled.Provided that the condition is met that the liquid passes through themacroporous gel, it is not necessary that the void volume of the supportmember be completely occupied by the macroporous gel.

[0025] The porous support member, or host, may be hydrophilic orhydrophobic and can be, for example, in the form of a membrane, achromatography bed, or a filtration bed. The support member provides themechanical strength to support the macroporous gel. The macroporous gelprovides a low resistance to hydraulic flow, enabling high flow rates tobe achieved with low reductions in pressure across the compositematerial. The macroporous gel also provides the separating function ofthe composite material in chromatographic and filtration applications.

[0026] A gel is a cross-linked polymer network swollen in a liquidmedium. The swelling liquid prevents the polymer network from collapsingand the network, in turn, retains the liquid.

[0027] Gels are typically obtained by polymerization of a monomer and apolyfunctional compound (a cross-linker), or by cross-linking across-linkable polymer, in a solvent which is a good solvent for theformed polymer network and which swells the polymer network. The polymerchains in such a network can be assumed to be uniformly distributedthroughout the whole volume of the network and the average distancebetween the chains, known as mesh size, is determined by thecross-linking density. As the concentration of the cross-linker isincreased, the density of cross-links in the gel also increases, whichleads to a smaller mesh size in the gel. The smaller mesh size resultsin a higher resistance to the flow of liquids through the gel. As theconcentration of the cross-linker is increased further, the constituentsof the gel begin to aggregate, which produces regions of high polymerdensity and regions of low polymer density in the gel. Such gels exhibitwhat has been called microheterogeneity. This aggregation normallycauses the gel to display a higher permeability to liquids, as the flowof liquids takes place primarily through the areas in the gel that havea lower polymer density. The low density areas of the gels are definedas draining regions while the higher density aggregates are callednon-draining regions. As the concentration of the cross-linker isincreased even further, leading to more cross-links, the gel can developregions in which there is essentially no polymer. These regions arereferred to as “macropores” in the present specification.

[0028] It is possible to compare the hydrodynamic (Darcy) permeabilityof a particular composite material of the invention with a referencematerial. The reference material is obtained by filling the pores of asupport member identical with that of the composite material with ahomogeneous gel of essentially the same chemical composition and thesimilar mass as the gel of the composite material, that is a gelcomposed of the same monomers formed in a good solvent, but cross-linkedonly to such an extent that the gel remains homogeneous and aggregationinto regions of high and low polymer density does not occur. Compositematerials having macroporous gels display hydrodynamic (Darcy)permeabilities that are at least one order of magnitude higher thanthose of the corresponding reference materials, and in some instancesthe permeabilities are more than two or even more than three orders ofmagnitude higher. In this specification, a composite material of theinvention whose hydrodynamic (Darcy) permeability is at least an orderof magnitude greater than that of the corresponding reference materialis said to have a permeability ratio greater than 10.

[0029] The permeability ratio is closely related to the size of themacropores in the composite material. For size-exclusion separationssuch as ultrafiltration, the permeability ratio can be fairly close to10. In other applications, for example adsorption, synthesis or cellgrowth, where larger macropores are used, the permeability ratio canreach, in some embodiments, values of 100 or greater, or even 1000 orgreater. In some instances it is possible to calculate the hydrodynamicpermeability of homogeneous gels, in accordance with the teachings ofMika A. M. and Childs R. F. Calculation of the hydrodynamic permeabilityof gels and gel-filled microporous membranes, Ind. Eng. Chem. Res., vol.40 (2001), p. 1694-1705, incorporated herein by reference. This dependsupon data for the particular gel polymer being available.

[0030] From the hydrodynamic permeability there can be derived thehydrodynamic radius, defined as the ratio of the pore volume to the porewetted surface area. It can be calculated from the hydrodynamic (Darcy)permeability using the Carman-Kozeny equation as given, for example, inthe book J. Happel and H. Brenner, Low Reynolds Numbers Hydrodynamics,Noordhof of Int. Publ., Leyden, 1973, p. 393, incorporated by referenceherein. It is necessary to assume a value for the Kozeny constant andfor the purpose of these calculations the inventors assume a value of 5.Composite materials of the invention, containing macroporous gels, arefound to have a hydrodynamic radius more than three times as high as thehydrodynamic radius of the corresponding reference material.

[0031] From the definition of the hydrodynamic permeability it can bederived that two composite materials of the same thickness will havehydrodynamic fluxes at the same pressure that will have the same ratioas their permeability ratio.

[0032] The size of macropores in the gel can be within a broad range,from a few nanometers to several hundred nanometers. Preferably, theporous gel constituent of the composite material has macropores ofaverage size between about 10 and about 3000 nm, has volume porositybetween 30 and 80% and a thickness equal to that of the porous supportmember. In some embodiments, the average size of the macropores ispreferably between 25 and 1500 nm, more preferably between 50 and 1000nm, and most preferably the average size of the macropores is about 700nm.

[0033] In the absence of a support member, the macroporous gels used inthe present invention may be non-self supporting, and they may change oreven lose their porosity when dried. By inserting the macroporous gelwithin a porous support member, mechanical strength is conferred uponthe macroporous gel. The utilization of macroporous gels creates acomposite material that permits larger molecules, such as biologicalmolecules, to enter the macropores and the solution containing suchmolecules to traverse the gel at a high flux.

[0034] By a “responsive composite material” is meant a compositematerial which comprises a macroporous gel whose pore-size can becontrolled by varying specific environmental conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is an environmental scanning electron microscope (ESEM)image of a macroporous poly(APTAC) gel;

[0036]FIG. 2 is an ESEM image of a macroporous poly(APTAC) gelincorporated into a support member in the form of a membrane;

[0037]FIG. 3 is a lysozyme adsorption curve of the membrane prepared inExample 3, below. The membrane volume is 0.467 ml;

[0038]FIG. 4 is a BSA adsorption curve of the membrane prepared inExample 8, below;

[0039]FIG. 5 is a graphical representation of the hydraulic radius as afunction of mass gain with photo- and thermally initiated porous gelcontaining membranes. Gel: poly(glycidyl methacrylate-co-ethylenediacrylate); solvents: dodecanol(DDC)/cyclohexanol(CHX) 9/91;

[0040]FIG. 6 is a graphical representation of the mass gain as afunction of total monomer concentration during the preparation ofcomposite membranes;

[0041]FIG. 7 s an AFM image of the surface of the AM610 membrane;(scanned area: 100 μm²);

[0042]FIG. 8 shows ESEM images of a nascent (top) and AM610 (bottom)surfaces; (magnification: 5000×);

[0043]FIG. 9 shows ESEM images of the surface of AM611 membrane;(magnification: top—5000×, bottom—3500×);

[0044]FIG. 10 shows ESEM images of membranes AM610 (top) and AM611(bottom); (magnification 5000×);

[0045]FIG. 11 is a lysozyme adsorption curve of the membrane prepared inExample 15. The membrane volume is 0.501 ml;

[0046]FIG. 12 is a lysozyme adsorption curve of the membrane prepared inExample 16. The membrane volume is 0.470 ml;

[0047]FIG. 13 is a lysozyme adsorption curve of the membrane prepared inExample 17. The membrane volume is 0.470 ml;

[0048]FIG. 14 is an ESEM image of a wet macroporous gel that is theproduct of Example 25;

[0049]FIG. 15 is an ESEM image of a wet microporous gel in a fibrousnon-woven support member that is the product of Example 26; and

[0050]FIG. 16 shows graphically results of using a multi-membrane stackof composite material of Example 22 in a protein (BSA) adsorption test.

[0051]FIG. 17 graphically displays the effect of monomer concentrationon the mass gain of composite materials.

[0052]FIG. 18 graphically displays the effect of ionic interactions onthe flux through a composite material at a pressure of 100 kPa.

[0053]FIG. 19 graphically displays changes in trans-membrane pressureand permeate conductivity as a function of salt concentration in thepermeate (A and B) and the changes of trans-membrane pressure as afunction of permeate conductivity (salt concentration) (C).

[0054]FIG. 20 shows the relationship between trans-membrane pressure,conductivity and absorbance for the HIgG ultrafiltration carried out inExample 39.

[0055]FIG. 21 shows the relationship between conductivity and absorbancefor the HSA ultrafiltration carried out in Example 39.

[0056]FIG. 22 shows the relationship between trans-membrane pressure,conductivity and absorbance for the HSA/HIgG ultrafiltration carried outin Example 39.

DETAILED DESCRIPTION OF THE INVENTION General Characteristics

[0057] Preferably, the macroporous gel is anchored within the supportmember. The term “anchored” is intended to mean that the gel is heldwithin the pores of the support member, but the term is not necessarilyrestricted to mean that the gel is chemically bound to the pores of thesupport member. The gel can be held by the physical constraint imposedupon it by enmeshing and intertwining with structural elements of thehost, without actually being chemically grafted to the host or supportmember, although in some embodiments, the macroporous gel may becomegrafted to the surface of the pores of the support member.

[0058] It will be appreciated that as the macropores are present in thegel that fills the pores of the support member, the macropores must besmaller than the pores of the support member. Consequently, the flowcharacteristics and separation characteristics of the composite materialare dependent on the characteristics of the macroporous gel, but arelargely independent of the characteristics of the porous support member,with the proviso, of course, that the size of the pores present in thesupport member is greater than the size of the macropores of the gel.The porosity of the composite material can be tailored by filling thesupport member with a gel whose porosity is primarily controlled by thenature and amounts of monomer or polymer, cross-linking agent, reactionsolvent, and porogen, if used. As pores of the support member are filledwith the same macroporous gel material, there is achieved a high degreeof consistency in properties of the composite material, and for aparticular support member these properties are determined largely, ifnot entirely, by the properties of the macroporous gel. The net resultis that the invention provides control over macropore-size, permeabilityand surface area of the composite materials.

[0059] The number of macropores in the composite material is notdictated by the number of pores in the support material. The number ofmacropores in the composite material can be much greater than the numberof pores in the support member, although the macropores are smaller thanthe pores in the support member. As mentioned above, the effect of thepore-size of the support material on the pore-size of the macroporousgel is generally quite negligible. An exception to this is found inthose cases where the support member has a large difference in pore-sizeand pore-size distribution, and where a macroporous gel having verysmall pore-sizes and a narrow range in pore-size distribution is sought.In these cases, large variations in the pore-size distribution of thesupport member are weakly reflected in the pore-size distribution of themacroporous gel. As such it is preferable to use a support member with asomewhat narrow pore-size range in these situations.

[0060] The properties of the composite materials can be tuned, byadjusting the average pore diameter of the macroporous gel. For somepurposes, for example ultrafiltration by means of size exclusion, smallpores may be required. For other purposes, for example use as a solidsupport for a chemical synthesis involving fast-kinetics, large poresmay be required. The size of the macropores is mainly dependent on thenature and concentration of the cross-linking agent, the nature or thesolvent or solvents in which the gel is formed, the amount of anypolymerization initiator or catalyst and, if present, the nature andconcentration of porogen.

[0061] Generally, as the concentration of cross-linking agent isincreased, the size of the macropores in the gel is also increased. Forexample, the molar ratio of polyfunctional compound(s) (cross-linkingagent) to monomer(s) may be in the range of from about 5:95 to about70:30, preferably in the range of from about 10:90 to about 50:50, andmore preferably in the range of from about 15:85 to about 45:55.

[0062] The components of the macroporous gel are introduced into thepores of the support member by means of a liquid vehicle, and solventselection for in situ polymerization or cross-linking plays a role inobtaining porous gels. Generally, the solvent or solvent mixture shoulddissolve monomers and polyfunctional compounds, or cross-linkablepolymers and cross-linking agents, over a wide range of concentrations.If the solvent is a good solvent for the gel polymer, porosity can onlybe introduced into the gel by cross-linking or porogen. If, however,there is present a solvent that is a thermodynamically poor solvent ornon-solvent, this solvent will act as a porogen. By combining solventsof different affinities to the gel polymer, from a good solvent througha poor solvent to a non-solvent, at different ratios, both porosity andpore dimensions can be altered. In general, the poorer the solvent orthe solvent mixture the higher the porosity and the sizes of macropores.Preferably, the solvent or solvent mixture for in situ polymerizationcontains poor solvent in the range from about 0% to about 100%, morepreferably from about 10% to about 90%. Examples of good solvents forpoly(2-acrylamido-2-methyl-1-propanesulfonic acid) are water andN,N-dimethylformamide. Examples of poor solvents include dioxane,hydrocarbons, esters, and ketones. An example of a good solvent forpoly(acrylamide) is water. Examples of poor solvents include dioxane,alcohols such as methanol, N,N-dimethylformamide, hydrocarbons, esters,and ketones. Preferably, the solvents used are miscible with water.

[0063] When the polymerization is carried out using a liquid vehiclethat contains non-solvents or poor solvents, the resulting structure isoften built of clusters of agglomerated microspheres that form the bodyof the macroporous gel. The pores in such materials consist of the voidslocated between clusters (macropores), voids between microspheres in theclusters (mesopores), and pores inside the microspheres themselves(micropores).

[0064] Porogens can be broadly described as pore generating additives.Examples of porogens that can be used in the gel-forming reactioninclude thermodynamically poor solvents or extractable polymers, forexample poly(ethyleneglycol), or surfactants, or salts. Porogens areknown in the art, and a person skilled can determine, using standardexperimental techniques and without exercise of any inventive faculty,which porogens are suitable to prepare macroporous gels for use in adesired composite material.

[0065] There is no simple way to predict accurately the structureparameters of porous gels obtained under given conditions, butqualitative rules are available to give some guidance. Generally, themechanism of porous gel formation via polymerization of one or moremonomers and cross-linkers involves, as a first step, an agglomerationof polymer chains to give nuclei. The polymerization continues both inthe nuclei and in the remaining solution to form microspheres which growin size by capturing newly precipitated nuclei and polymers from thesolution. At some point, the microspheres become interconnected witheach other in large clusters that form the body of the macroporous gel.The poorer the solvent quality the faster nucleation occurs during thegel-forming process. If the number of nuclei formed is very large, as inthe case of high concentration of a polymerization initiator, smallerpores may be expected. If, however, the number of nuclei is smaller andthe reaction kinetics is such that the nuclei can grow larger, largepores are formed in the gel. High concentration of a cross-linkerusually causes early nucleation. The nuclei, however, may be too highlycross-linked to be able to swell with the monomers, grow and coalesce inclusters. This may result in very small pores. Because of the differentways that the polymerization may proceed and the polymerizationconditions may affect the gel porous structure, a large variety ofstructures can be obtained but conditions for each of the structuresneed to be determined experimentally.

Separations with the Composite Material

[0066] In some embodiments of the invention the composite material isused as a separating medium, for example in filtration operations wherea liquid to be filtered is passed through the composite membrane andseparation of one or more components from the liquid is effected by sizeexclusion in an uncharged macroporous gel. The separation can further beenhanced by the Donnan exclusion of charged molecules by use of acharged macroporous gel. If the macroporous gel contains a fixed chargeand the charge of the solutes can be appropriately adjusted, the solutescan be separated even against their size gradient. For example with asolution containing a mixture of proteins, if a pH value is selected forwhich one of the proteins in the mixture is at its isoelectric pointwhile the other proteins retain charge of the same sign as the membranecharge, the other proteins can be held back in the retentate because ofthe charge repulsion with the membrane. By tailoring the conditions forfractionation, good selectivity, even for proteins of the same size, canbe obtained.

[0067] Separation can also be achieved by the presence of reactivefunctional groups in the macroporous gel. These functional groups can beused to bear a ligand or other specific binding site that has anaffinity to a molecule or ion, including a biomolecule or biomolecularion. When a liquid containing the particular molecule or ion is passedthrough the composite material the ligand or specific binding siteinteracts with the molecule or ion enough to adsorb it. In some cases itis possible to subsequently desorb the captured molecule or ion when theenvironment around the composite material is subsequently altered, forexample by changing the nature of the solvent passed through themacropores of the gel. The binding sites can also include chargedgroups.

Composition of the Macroporous Gels

[0068] The macroporous gels can be formed through the in-situ reactionof one or more polymerisable monomers with one or more cross-linkers, orof one or more cross-linkable polymers with one or more cross-linker toform a cross-linked gel that has macropores of a suitable size. Suitablepolymerisable monomers include monomers containing vinyl or acrylgroups. For Donnan exclusion, there can be used vinyl or acryl monomerscontaining at least one polar and/or ionic functional group, orfunctional group that can be converted into ionic group. For biologicalaffinity there can be used vinyl or acryl monomers containing at leastone reactive functional group. Preferred polymerisable monomers includeacrylamide, 2-acryloxyethyltrimethylammonium chloride,N-acryloxysuccinimide, N-acryloyltris(hydroxymethyl)methylamine,2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, butyl acrylate and methacrylate, N,N-diethylacrylamide,N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate andmethacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide,N,N-dimethylacrylamide, n-dodecyl acrylate, n-dodecyl methacrylate,dodecyl methacrylamide, ethyl methacrylate, 2-(2-ethoxyethoxy)ethylacrylate and methacrylate, 2,3-dihydroxypropyl acrylate andmethacrylate, glycidyl acrylate and methacrylate, n-heptyl acrylate andmethacrylate, 1-hexadecyl acrylate and methacrylate, 2-hydroxyethylacrylate and methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl acrylate and methacrylate, methacrylamide, methacrylicanhydride, methacryloxyethyltrimethylammonium chloride,2-(2-methoxy)ethyl acrylate and methacrylate, octadecyl acrylamide,octylacrylamide, octyl methacrylate, propyl acrylate and methacrylate,N-iso-propylacrylamide, stearyl acrylate, styrene, 4-vinylpyridine,vinylsulfonic acid, N-vinyl-2-pyrrodinone. Particularly preferredmonomers include dimethyldiallylammonium chloride,acrylamido-2-methyl-1-propanesulfonic acid (AMPS),(3-acrylamidopropyl)trimethylammonium chloride (APTAC), acrylamide,methacrylic acid (MAA), acrylic acid (AA), 4-styrenesulfonic acid andits salts, acrylamide, glycidyl methacrylate, diallylamine,anddiallylammonium chloride.

[0069] The crosslinker may be, for example, a compound containing atleast two vinyl or acryl groups. Examples of crosslinkers includebisacrylamidoacetic acid, 2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate anddimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanedioldiacrylate and dimethacrylate, 1,10-dodecanediol diacrylate anddimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,2,2-dimethylpropanediol diacrylate and dimethacrylate, dipentaerythritolpentaacrylate, dipropylene glycol diacrylate and dimethacrylate,N,N-dodecamethylenebisacrylamide, divinylbenzene, glyceroltrimethacrylate, glycerol tris(acryloxypropyl) ether,N,N′-hexamethylenebisacrylamide, N,N′-octamethylenebisacrylamide,1,5-pentanediol diacrylate and dimethacrylate, 1,3-phenylenediacrylate,poly(ethylene glycol)diacrylate and dimethacrylate,poly(propylene)diacrylate and dimethacrylate, triethylene glycoldiacrylate and dimethacrylate, triethylene glycol divinyl ether,tripropylene glycol diacrylate or dimethacrylate, diallyl diglycolcarbonate, poly(ethylene glycol) divinyl ether,N,N′-dimethacryloylpiperazine, divinyl glycol, ethylene glycoldiacrylate, ethylene glycol dimethacrylate, N,N′-methylenebisacrylamide,1,1,1-trimethylolethane trimethacrylate, 1,1,1-trimethylolpropanetriacrylate, 1,1,1-trimethylolpropane trimethacrylate, vinyl acrylate,1,6-hexanediol diacrylate and dimethacrylate, 1,3-butylene glycoldiacrylate and dimethacrylate, alkoxylated cyclohexane dimethanoldicarylate, alkoxylated hexanediol diacrylate, alkoxylated neopentylglycol diacrylate, aromatic dimethacrylate, caprolacone modifiedneopentylglycol hydroxypivalate diacrylate, cyclohexane dimethanoldiacrylate and dimethacrylate, ethoxylated bisphenol diacrylate anddimethacrylate, neopentyl glycol diacrylate and dimethacrylate,ethoxylated trimethylolpropane triarylate, propoxylatedtrimethylolpropane triacrylate, propoxylated glyceryl triacrylate,pentaerythritol triacrylate, tris (2-hydroxy ethyl)isocyanuratetriacrylate, di-trimethylolpropane tetraacrylate, dipentaerythritolpentaacrylate,ethoxylated pentaerythritol tetraacrylate, pentaacrylateester, pentaerythritol tetraacrylate, and caprolactone modifieddipentaerythritol hexaacrylate. Particularly preferred cross-linkingagents include N,N′-methylenebisacrylamide, diethylene glycol diacrylateand dimethacrylate, trimethylolpropane triacrylate, ethylene glycoldiacrylate and dimethacrylate, tetra(ethylene glycol)diacrylate,1,6-hexanediol diacrylate, divinylbenzene, poly(ethylene glycol)diacrylate.

[0070] The concentration of monomer in the macroporous gel can have aneffect on the resiliency of the macroporous gel prepared. A low monomerconcentration can lead to a macroporous gel that is non-self supporting.Such non-self supporting gels might be advantageous as adsorbents, asthey could lead to gels having greater adsorption capacity. In someembodiments, the monomer concentration is 60% or less, for example about60, 50, 40, 30, 20, 10 or 5%.

[0071] When a cross-linkable polymer is used, it can be dissolved andreacted in-situ in the support with a cross-linking agent to form themacroporous gel. Suitable cross-linkable polymers includepoly(ethyleneimine), poly(4-vinylpyridine), poly(vinylbenzyl chloride),poly(diallylammonium chloride), poly(glycidyl methacrylate),poly(allylamine), copolymers of vinylpyridine anddimethyldiallylammonium chloride, copolymers of vinylpyridine,dimethyladiallylammonium chloride, or(3-acrylamidopropyl)trimethylammonium chloride with glycidyl acrylate ormethacrylate, of which poly(ethyleneimine), poly(diallylammoniumchloride), and poly(glycidyl methacrylate) are preferred. Use ofcross-linkable polymers instead of monomers can, in some instances,require a decrease in the concentration of cross-linking agent. In orderto retain the large size of the pores in the gel with a lowercross-linking agent concentration, a porogen can be added to the mixtureused to prepare the macroporous gel.

[0072] The cross-linking agent for reaction with the cross-linkablepolymer is selected from molecules containing two or more reactivegroups that can react with an atom or group of atoms in the polymer tobe cross-linked, such as epoxy groups or alkyl/aryl halides that canreact with nitrogen atoms of polyamines, or amine groups that can reactwith alkyl/aryl halides or epoxy groups of glycidyl-group-containingpolymers to be in situ-cross-linked. Suitable cross-linkers includeethylene glycol diglycidyl ether, poly(propylene glycol) diglycidylether, 1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane,1,6-dibromohexane, α,α′-dibromo-p-xylene, α,α′-dichloro-p-xylene,1,4-dibromo-2-butene, piperazine, 1,4-diazabicyclo[2.2.2]octane,1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane,1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane.

[0073] It is also possible to modify polymers containing reactive groupssuch as an amino, hydroxyl, carboxylic acid, carboxylic acid ester, orepoxy groups with reagents to introduce vinyl groups that can besubsequently polymerized by treatment with a polymerization initiator toform a macroporous gel. Examples of suitable vinyl groups that can beintroduced include vinylbenzene derivatives, allyl derivatives, acrolyland methacrolyl derivatives. The cross-linking of these vinylsubstituted polymers can in some instances be facilitated by theintroduction of further monomers such as acrylamide, N-vinylpyrrolidone,acrylic and methacrylic acids and their salts.

[0074] Macromonomers can also be used as monomers or as cross-linkingagents. Macromonomers can be polymers or oligomers that have one(monofunctional) or more (cross-linking agent) reactive groups, often atthe ends, which enable them to act as a monomer or a cross-linker. Formonomers, each macromonomer molecule is attached to the main chain ofthe final polymer by reaction of only one monomeric unit in themacromonomer molecule. Examples of macromonomers include poly(ethyleneglycol)acrylate and poly(ethylene glycol)methacrylate, while examples ofpolyfunctional macromonomers include poly(ethylene glycol)diacrylate andpoly(ethylene glycol)dimethacrylate. Macromonomers preferably havemolecular weights of about 200 Da or greater.

[0075] Many macroporous gels can be prepared, including neutralhydrogels, charged hydrogels, polyelectrolyte gels, hydrophobic gels,and neutral and functional gels.

[0076] If the gel selected is a neutral hydrogel or a charged hydrogelfor which water is the swelling liquid medium, the resulting supportedmacroporous gel is normally quite hydrophilic. Hydrophilic compositematerials are preferred as they provide better flow characteristics andimpart anti-fouling tendencies to the membranes. Examples of suitablehydrogels include cross-linked gels of poly(vinyl alcohol),poly(acrylamide), poly(isopropylacrylamide), poly(vinylpyrrolidone),poly(hydroxymethyl acrylate), poly(ethylene oxide), copolymers ofacrylic acid or methacrylic acid with acrylamide, isopropylacrylamide,or vinylpyrrolidone, copolymers of acrylamide-2-methyl-1-propanesulfonicacid with acrylamide, isopropylacrylamide, or vinylpyrrolidone,copolymers of (3-acrylamidopropyl) trimethylammonium chloride withacrylamide, isopropylacrylamide, or N-vinylpyrrolidone, copolymers ofdiallyldimethylammonium chloride with acrylamide, isopropylacrylamide,or vinylpyrrolidone. Preferred hydrogels include cross-linked poly(vinylalcohol), poly(acrylamide), poly(isopropylacrylamide) andpol(vinylpyrrolidone) and cross-linked copolymers of neutral monomerssuch as acrylamide or N-vinylpyrrolidone with charged monomers such asacrylamide-2-methyl-1-propanesulfonic acid or diallyldimethylammoniumchloride.

[0077] The macroporous gels can be selected to comprisepolyelectrolytes. Like the charged hydrogels, polyelectrolyte gels givehydrophilic composite material, and they also carry a charge. Thepolyelectrolyte gel can be selected, for example, from cross-linkedpoly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts,poly(styrenesulfonic acid) and its salts, poly(vinylsulfonic acid) andits salts, poly(alginic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium)salts, poly(vinylbenzyl-N-trimethylammonium) salts, poly(ethyleneimine)and its salts. Preferred charged gels include cross-linked poly(acrylicacid) and its salts, poly(methacrylic acid) and its salts,poly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, and poly(4-vinylpyridinium) salts.

[0078] One of the differences between charged gels and polyelectrolytegels is that the repeating monomer in the polyelectrolyte gel bears acharge, while in the charged gel, the charge is found in aco-polymerized unit that is randomly distributed through the polymer.The monomer used to form the polyelectrolyte gel or the co-polymer inthe charged gel that bears a charge usually has a charge bearing group,but it can also be a non-charge-bearing group that can become charged ina post-gelation process (e.g. quaternization of nitrogen bearinggroups). Examples of polymers that can become charged includepoly(4-vinylpyridine) which can be quaternized with various alkyl andalkylaryl halides. Suitable alkyl halides include those having up to 8carbon atoms, for example methyl iodide, ethyl bromide, butyl bromide,and propyl bromide. Suitable alkylaryl halides include benzyl halides,especially benzyl chloride and benzyl bromide. Another polymer that canbecome charged is poly(vinylbenzyl chloride), which can be quaternizedwith various amines, for example, lower alkylamines or aromatic aminessuch as triethylamine, pyridine, azabicyclo[2.2.2]octane,N-methylpyrrolidine, and N-methylpiperidine, and lowerhydroxyalkylamines, for example triethanolamine. Yet another polymerthat can become charged is poly(glycidyl methacrylate) or poly(glycidylacrylate), which can react with various amines, for example loweralkylamines such as diethylamine and triethylamine,azabicyclo[2.2.2]octane, N-methylpyrrolidine, and N-methylpiperidine.Alternatively, glycidyl moieties can be converted to sulfonic acidgroups by reaction with, for example alkali metal sulfites such assodium sulfite. A person skilled in the art will appreciate that thereare other polymers that are, or can be rendered, charge-bearing.

[0079] The macroporous gel can be selected to comprise hydrophobicmonomers to permit separations in organic solvents, for examplehydrocarbons, especially liquid paraffins such as hexanes. Hydrophobicmonomers, such as styrene and its derivatives, for example an alkylsubstituted styrene derivative such as para-tertbutyl styrene, can beused to prepare hydrophobic macroporous gels. Copolymers of thesemonomers can be used.

[0080] A macroporous gel comprising hydrophobic monomers can be used tocapture molecules from fluids passing through the pores by hydrophobicinteractions.

[0081] As stated above, the macroporous gels can also be selected tocomprise reactive functional groups that can be used to attach ligandsor other specific binding sites. These functional macroporous gels canbe prepared from cross-linked polymers bearing functional groups, forexample epoxy, anhydride, azide, reactive halogen, or acid chloridegroups, that can be used to attach the ligands or other specific bindingsites. Examples include cross-linked poly(glycidyl methacrylate),poly(acrylamidoxime), poly(acrylic anhydride), poly(azelaic anhydride),poly(maleic anhydride), poly(hydrazide), poly(acryloyl chloride),poly(2-bromoethyl methacrylate),poly(vinyl methyl ketone). Functionalitythat can be introduced can take the form of antibodies or fragments ofantibodies, or alternatively, chemical mimics such as dyes. Functionalgels are attractive in biomolecule purifications or separations, as theycan offer preferential binding to certain molecules by binding to activesites, while being non-reactive to other molecules, even when there isno significant difference in size between the molecules, examples beingaffinity ligands selected to bind with some proteins but not others.Affinity ligands that can be attached to porous gels via reactive groupsinclude amino acid ligands such as L-phenylalanine, tryptophan, orL-histidine to separate γ-globulins and immunoglobulins, antigen andantibody ligands such as monoclonal antibodies, protein A, recombinantprotein A, protein G, or recombinant protein G to separateimmunoglobulins from different media, dye ligands such as cibaron blueor active red to separate albumins and various enzymes, metal affinityligands such as complexes of iminodiacetic acid (IDA) ligand with Cu²⁺,Ni²⁺, Zn²⁺, or Co²⁺ to separate various proteins such as histidine,lysozyme, or tryptophan from various media.

Responsive Macroporous Gels

[0082] Polymers that change their conformation in response to changes inenvironmental conditions are known. By incorporating the properties ofsuch polymers in the macroporous gel, a composite material with dynamicpore-size is obtained. These composite materials having responsivecharacteristics are substantially the same as the composite materialsdescribed above, except that at least one of the monomers or polymersthat form the macroporous gel has a chemical structure which facilitateschanges in pore-size.

[0083] The changes in the pore-size of the macroporous gel are due tothe physical relationship between the support member and the macroporousgel. The composite material can be described as having three distinctzones: (a) the support member, which ideally does not change shape, (b)the incorporated macroporous gel that “fills” the pores of the supportmember, and (c) the volume within the macropores of the gel, whichvolume is filled with water or a solvent and in which is found verylittle or no gel polymer. Under pressure, hydraulic flow occurs throughthe macropores of the gel, and the flux through the composite materialis related to the number of pores in the macroporous gel, the radius ofthese pores, and the tortuosity of the path of the pores in themacroporous gel through the composite material.

[0084] As the degree of swelling of the macroporous gel is changed by anenvironmental stimulus, the total volume occupied by the macroporous gelis constrained by the fixed total volume defined by the support member.As the overall volume of the macroporous gel is constrained by thesupport member, by necessity the volume fraction of the gel expands intothe area defined by macropores in the gel. As the number of macroporesand their tortuosity remain essentially constant with the change involume fraction of the macroporous gel, the diameter or radius of themacropores themselves must change. If the macroporous or structured gelwere unconfined, the environmentally induced changes would cause thetotal volume of the swollen gel to change. As such it would not followin this unconfined case that the changes would result in a controllablechange in pore-size of the macroporous gel.

[0085] The reason behind the change in volume of the macroporous gel isrelated to interactions between the polymer structures that form thegels, or the interactions between the polymer chains and the solvents orsolutes present in the solvent that diffuse into the gel. The changes inthe volume occupied by the gel are linked to the conformation adopted bythe polymer chains that form the macroporous gels. The natural tendencyof the polymer chains is to coil around themselves, which leads to a gelhaving a smaller volume. If the polymer chains within the gel can bemanipulated to uncoil and form a more rigid backbone, the overall volumeof the gel will increase. It is thus this coiling/uncoiling which isaffected by the environmental stimuli that are applied to the responsivecomposite material.

[0086] The volume changes of the pores can either be “continuous” or“discontinuous”. A continuous volume change takes place over arelatively large change in the triggering environmental condition andwhere there exists at least one stable volume near the transitionbetween the swollen and collapsed state. Preferably, a continuous volumechange will go through a multitude of stable transition volumes betweenthe swollen and the collapsed state. A discontinuous volume change ingels is characterised by the reversible transition from swollen tocollapsed state taking place over an extremely small change in thetriggering environmental condition, for example, less than 0.1 pH unitor 0.1 degree Celsius. Gels exhibiting discontinuous volume change arecalled “phase-transition” gels and systems or devices with such gels areoften called “chemical valves”. Preferably, the responsive macroporousgels according to this embodiment of the invention undergo a“continuous” volume change through discrete stable volumes that can beutilized to control the pore-size of the gel.

[0087] Of the environmental stimuli that can be used to change thepore-size in the responsive macroporous, mention is made of pH, specificions, ionic strength, temperature, light, electric fields, and magneticfields. The effect of each stimulus, and examples of monomers that reactto such a stimulus, will be described in more detail below.

[0088] One stimulus that can be utilised to change the pore-size ofresponsive macroporous gel is the pH of the solution being passedthrough the pores of the gel. A change in the pH of the solution willaffect the pore-size of the gel if the gel comprises weak acids or weakbases. In such cases, the natural tendency of the polymer chain withinthe gel to coil around itself will be balanced by the repulsion betweenthe charged groups (weak acidic or basic groups) along the length of thepolymer chain. Variations in the amount of charge along the chain causelarge changes in conformation of the polymer chain, which in turn causeschanges in the volume occupied by the gel. Changes in the pH of thesolution are effective at controlling the amount of repulsion along thepolymer chain, as they change the degree of ionisation of the chargedgroups. A gel comprising weak acid groups becomes less ionised as the pHis lowered and the gel contracts. Conversely, a weak base becomes moreionised as the pH is lowered and the chain elongates or stretches togive a swollen gel.

[0089] Examples of monomers that have weak acid functionality includeacrylic acid, methacrylic acid, itaconic acid, 4-vinylbenzoic acid,bisacrylamidoacetic acid, and bis(2-methacryloxyethyl) phosphate.Examples of monomers that have weak base functionality include2-aminoethyl methacrylate hydrochloride, N-(3-aminopropyl)methacrylamidehydrochloride, 2-(tert-butylamino)ethyl methacrylate, diallylamine,2-(N,N-diethylamino)ethyl methacrylate, 2-(N,N-dimethylamino)ethylacrylate, 2-(N,N-dimethylamino)ethyl methacrylate, 1-vinylimidazole, and4-vinylpyridine. Glycidyl methacrylate derivatizedhyaluronate-hydroxyethyl acrylate based hydrogels can also be used toprepare composite materials that are pH responsive [Inukai M., Jin Y.,Yomota C., Yonese M., Chem. Pharm. Bull., (2000), 48:850-854; which ishereby incorporated by reference].

[0090] Variations in pH have little effect on the degree of ionisationof strong acids and bases, and as such, only drastic variations in pHcan effect pore-size changes in gels comprising these functionalities.

[0091] Another stimulus that can be utilised for changing the pore-sizeof a responsive macroporous gel is the salt concentration of thesolution being passed through the pores of the gel. Similarly tovariations in pH, variations in salt concentration will effect pore-sizevariations in macroporous gels that comprise weak acidic or weak basicgroups. The reason for the changes in pore-size, however, does differslightly. The addition of an ionic solute has the ability to shield thecharged groups found on the polymer chain in the gel by the formation ofion-pairs. This lessens the coulombic repulsion between the adjacentcharged groups, which allows the chain to relax into a coiledconformation. An increase in salt concentration will shield both a weakacid group and a weak base group. Therefore, when the salt concentrationis increased, for example by adding a concentrated salt solution to thebulk solution being passed through the composite material, the shieldingeffect of the additional ions leads to an increase in pore size.Alternatively, a decrease in salt concentration, such as obtained bydiluting the bulk solution being passed through the composite material,will lead to less shielding and a smaller pore size.

[0092] Changes in salt concentration can also be used with macroporousgels that comprise strong acid groups and strong basic groups, as thesegroups are also shielded by the presence of free ionic species.

[0093] Examples of monomers that bear weak acid or base groups arelisted above. Examples of monomers that have strong acid functionalityinclude 2-acrylamido-2-methyl-1-propanesulfonic acid, sodium2-methyl-2-propene-1-sulfonate, sodium styrenesulfonate, and sodiumvinylsulfonate. Examples of monomers that have strong basicfunctionality include 2-acryloxyethyltrimethylammonium chloride,diallyldimethylammonium chloride, methacrylamidopropyltrimethylammoniumchloride, and 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride.Ionic functionality (weak/strong acids and bases) may also be introducedin macroporous gels that do not originally bear charged functionalitiesbut that bear instead reactive groups that can be converted into ionicor ionisable moieties in a post-polymerization treatment. Suitablemonomers with reactive groups include acrylic anhydride, allyl glycidylether, bromostyrene, chloromethylstyrene, chlorostyrenes, glycidylacrylate, glycidyl methacrylate, 4-hydroxybutyl methacrylate,2-hydroxyethyl acrylate, methacryloyl chloride. For example, amacroporous gel comprising a glycidyl acrylate or methacrylate group canbe treated with diethylamine to introduce weak base functionality orwith sodium sulfite in an iso-propanol/water mixture to introduce strongacid (sulfonic acid) functionality.

[0094] Another stimulus that can be used to change the pore-size of aresponsive macroporous gel is the temperature of the gel. Variousmethods are available for changing the temperature of the macroporousgel, one of which includes changing the temperature of a liquid flowingthrough the pores of the macroporous gel. While the change in overallgel volume for temperature dependant gels is again due to the control ofthe coiling or uncoiling of the polymer chains that form the gel, thecontraction or expansion of the gel is not linked to the presence ofcharged groups on the polymer chain. For temperature dependant gels, theamount solvation of the polymer chain controls the conformation of thepolymer chain. At lower temperatures the chains are solvated, whichallows an elongated conformation of the polymer chain. As thetemperature is increased, an entropic desolvation takes place causingthe chains to coil and contract. Therefore, increases in temperaturelead to larger pore sizes in the gel while decreases in temperature leadto'smaller pore sizes.

[0095] Macroporous gels that comprise hydrophobic monomers are mostsuitable for use in temperature dependant systems, as solvation effectsare markedly observed for polymers that have hydrophobic functionality.Examples of monomers that have hydrophobic functionality includeN-(propyl)acrylamide, N-(tert-butyl)acrylamide, butyl acrylates, decylacrylates, decyl methacrylates, 2-ethylbutyl methacrylate, n-heptylacrylate, n-heptyl methacrylate, 1-hexadecyl acrylate, 1-hexadecylmethacrylate, n-hexyl acrylate, n-hexyl methacrylate, andN-(n-octadecyl)acrylamide. Gels displaying thermal response can also beprepared from sulphated hyaluronic acid-based gels.(see Barbucci R.,Rappuoli R., Borzacchiello A., Ambrosio L., J. Biomater. Sci.-Polym.Ed., (2000), 11:383-399), incorporated herein by reference.

[0096] Light is another stimulus that can be used to change thepore-size of the responsive macroporous gel. Light induced changes aredue to photoisomerizations in the backbone or the side-chains of thepolymer chains that form the gel. These photoisomerizations cause achange in either the conformation and local dipole moment, or in thedegree of ionisation through light induced electron transfer reactions.One type of monomer that is suitable for use in light controlled systemscomprises unsaturated functionalities than can undergo a trans-cisisomerization on irradiation. Examples of photoresponsive monomers thatgo through cis-trans conformation and dipole changes include4-(4-oxy-4′-cyanoazobenzene)but-1-yl methacrylate,6-(4-oxy-4′-cyanoazobenzene)hex-1-yl methacrylate,8-(4-oxy-4′-cyanoazobenzene)oct-1-yl methacrylate,4-[ω-methacryloyloxyoligo(ethyleneglycol)]-4′-cyanoazobenzene,4-methacryloyloxy-4′-{2-cyano-3-oxy-3-[ω-methoxyoligo(ethyleneglycol)]prop-1-en-1-yl}azobenzene,and methacrylate monomers containing a mesogenic group and aphotochromic para-nitroazobenzene group. It is also possible toincorporate the photoresponsive moeity in the crosslinker instead of themonomer. Examples of photoresponsive crosslinkers that go throughcis-trans conformation and dipole changes include4,4′-Divinylazobenzene,N,N′-bis(β-styrylsulfonyl)-4,4′-diaminoazobenzene,4,4′-bis(methacryloylamino)azobenzene, 4,4′-dimethacryloylazobenzene,and bis((methacryloyloxy)methyl)spirobenzopyran.

[0097] The pore-size of the gel can also be altered by subjecting themacroporous gel to an electric field or to an electrical current. Theresponse of the gel to electrochemical current changes is closelyrelated to the pH systems described above. This close relationship isdue to the fact that the passage of an electrochemical current throughan aqueous system causes a “water splitting” reaction, which reactionleads to changes in the pH of the aqueous system. Electrical current canbe passed through a composite material of the invention e.g. by placingan electrode at either end of the composite material. When currentdifferential is applied to the electrodes, water molecules will separateand concentrations of H⁺ and HO⁻ will increase at their respectiveelectrodes. As described earlier, changes in pH can be used to controlthe pore-size of macroporous gels that comprise weak acid or weak basefunctionalities, which control is linked to the relationship between theionisation of these functionalities and the coiling/uncoiling of thepolymer chains that form the gel. Examples of weak acidic and weak basicmonomers are given above.

[0098] Changes in gel volume due to fluctuations in an electrical fieldhave been previously observed, such as in Murdan S., J. Control.Release, (2003), 92:1-17; and in Jensen M., Hansen P. B., Murdan S.,Frokjaer S., Florence A. T., Eur. J. Pharm. Sci., (2002), 15:139-148,which are hereby incorporated by reference. While the exact processthrough which the gel volume is changed by the application of anelectrical field is not yet well defined, the volume change itself iswell documented. Chondroitin 4-sulphate (CS) is an example of a monomerthat is responsive to electrical field fluctuations.

[0099] In some embodiments, the various stimuli response systems can becombined to offer gels that respond to more than one stimulus. Anexample of such a combined system can be prepared by combining a chargedpolymer (weak/strong acid or base) with a hydrophobic monomer. Themacroporous gel resulting from such a combination will display responsesto changes in salt concentration, changes in solution pH (when weakacids or bases are used), and changes in temperature. When combiningdifferent monomers, it is possible that the responsiveness of the gel toa single of the stimuli will be diminished, as the concentration of themonomer that responds to that particular stimulus will be lowered in thegel.

[0100] The magnitude of the response expressed by the macroporous gels,when various stimuli are applied to the gel, depends many differentvariables, a few of which are discussed below:

[0101] The responsiveness of the macroporous gel is dependent on theconcentration of the crosslinking agent. Generally, as the concentrationof cross-linking agent is increased, the size of the macropores in theresponsive gel is also increased, but the range of pore-size changes isdecreased. This relationship is fairly straightforward, as a higherconcentration of crosslinks within the gel will limit the amount ofcoiling and uncoiling that will be available to the responsive gel. Themolar ratio of crosslinking agent(s) to monomer(s) may be in the rangeof from about 5:95 to about 40:60, preferably in the range of from about7.5:92.5 to about 10:90, and more preferably in the range of from about10:90 to about 25:75.

[0102] Certain stimuli naturally evoke a broader range of response inthe gel, as they more effectively affect the conformation of the polymerchains that form the gel. For example, variations in pH or temperatureevoke a strong response from the appropriate macroporous gels, whilechanges in salt concentrations and light intensity evoke a slightlysmaller response.

[0103] The concentration of the responsive monomer in the gel alsoaffects the level of response demonstrated by the gel. Preferably, theresponsive macroporous gels are composed of one or more responsivemonomers and of one or more neutral monomers. The presence of a neutralmonomer is important in those systems that have a very strong responseto changes in the environmental conditions, as such systems oftendisplay discontinuous responses in pore-size (valve-effects). Additionof a neutral monomer attenuates the response, permitting a morecontrolled change in pore-size. Preferably, the molar ratio of theneutral monomers to the molar ratio of responsive monomers in theresponsive macroporous gel is in the range from 5:95 to 95:5, morepreferably in the range from 25:75 to 75:25, and more preferably in therange from 40:60 to 60:40. Suitable neutral monomers include acrylamide,N-acryloylmorpholine, N-acryloxysuccinimide,2-acrylamido-2-(hydroxymethyl)-1,3-propanediol, N,N-diethylacrylamide,N,N-dimethylacrylamide, 2-(2-ethoxyethoxy)ethyl acrylate, 2-ethoxyethylmethacrylate, 2,3-dihydroxypropyl methacrylate, 2-hydroxyethylmethacrylate, N-(2-hydroxypropyl)methacrylamide, hydroxypropylmethacrylate, methacrylamide,N-[tris(hydroxymethyl)methyl]-1-methacrylamide, N-methylmethacrylamide,N-methyl-N-vinylacetamide, poly(ethylene glycol) monomethacrylate,N-iso-propylacrylamide, N-vinyl-2-pyrrolidone.

Porous Support Member

[0104] A variety of materials can be used to form the support member;however, apart from materials such as cellulose and some of itsderivatives, most of these materials are strongly or relativelyhydrophobic. Hydrophobic filtration membranes are not usually desiredfor use with aqueous systems, as they can lead to higher membranefouling tendencies. The more inert and cheaper polymers such aspolyolefins, for example (poly(ethylene), poly(propylene)poly(vinylidene difluoride)) can be used to make microporous membranes,but these materials are very hydrophobic. In some embodiments of thepresent invention, the hydrophobicity of the support member does notaffect the degree of fouling experienced by the composite material asthe flow of liquid through the composite material takes place primarilyin the macropores of the gel.

[0105] In some embodiments, the porous support member is made ofpolymeric material and contains pores of average size between about 0.1and about 25 μm, and a volume porosity between 40 and 90%. Many poroussubstrates or membranes can be used as the support member but thesupport is preferably a polymeric material, and it is more preferably apolyolefin, which, while hydrophobic, is available at low cost. Extendedpolyolefin membranes made by thermally induced phase separation (TIPS),or non-solvent induced phase separation are mentioned. Hydrophilicsupports can also be used, including natural polymers such as celluloseand its derivatives. Examples of suitable supports include SUPOR®polyethersulfone membranes manufactured by Pall Corporation,Cole-Parmer® Teflon® membranes, Cole-Parmer® nylon membranes, celluloseester membranes manufactured by Gelman Sciences, Whatman® filter andpapers.

[0106] In some other embodiments the porous support is composed of wovenor non-woven fibrous material, for example a polyolefin such aspolypropylene. An example of a polypropylene non-woven material iscommercially available as TR2611A from Hollingsworth and Vose Company.Such fibrous woven or non-woven support members can have pore sizeslarger than the TIPS support members, in some instances up to about 75μm. The larger pores in the support member permit formation of compositematerials having larger macropores in the macroporous gel. Compositematerials with larger macropores can be used, for example, as supportson which cell growth can be carried out. Non-polymeric support memberscan also be used, such as ceramic-based supports. The porous supportmember can take various shapes and sizes.

[0107] In some embodiments, the support member is in the form of amembrane that has a thickness of from about 10 to about 2000 μm, morepreferably from 10 to 1000 μm, and most preferably from 10 to 500 μm. Inother embodiments, multiple porous support units can be combined, forexample, by stacking. In one embodiment, a stack of porous supportmembranes, for example from 2 to 10 membranes, can be assembled beforethe macroporous gel is formed within the void of the porous support. Inanother embodiment, single support member units are used to formcomposite material membranes, which are then stacked before use.

Preparation of Composite Materials

[0108] The composite materials of the invention can be prepared bysimple, single step methods. These methods can, in some instances, usewater or other benign solvents, such as methanol, as the reactionsolvent. The methods also have the benefit of using rapid processes thatlead to easier and continuous manufacturing possibilities. The compositematerial is also potentially cheap.

[0109] The composite materials of the invention can be prepared, forexample, by mixing one or more monomers, one or more polymers, ormixtures thereof, one or more cross-linking agents, optionally one ormore initiators and optionally one or more porogens, in one or moresuitable solvents. The solution produced is preferably homogeneous, buta slightly heterogeneous solution can be used. The mixture is thenintroduced into a suitable porous support, where a gel forming reactiontakes place. Suitable solvents for the gel forming reaction include, forexample, water, dioxane, dimethylsulfoxide (DMSO), dimethylformamide(DMF), acetone, ethanol, N-methylpyrrolidone (NMP), tetrahydrofuran(THF), ethyl acetate, acetonitrile, toluene, xylenes, hexane,N-methylacetamide, propanol, and methanol. It is preferable to usesolvents that have a higher boiling point, as these solvents reduceflammability and facilitate manufacture. It is also preferable that thesolvents have a low toxicity, and they can be readily disposed of afteruse. An example of such a solvent is dipropyleneglycol monomethyl ether(DPM).

[0110] In some embodiments, it is possible to use dibasic esters (estersof a mixture of dibasic acids) as the solvent. Dibasic esters (DBEs) areespecially suitable for preparing gels based on polyacrylamide monomers.This solvent system has an unexpected characteristic in that it ispoorly soluble in water, which differs from the other solvents usedwhich are essentially completely water miscible. While water misciblesolvents offer advantages in terms of solvent removal after fabrication,water immiscible solvents such as DBE's are good replacements, incertain cases, for solvents such as dioxane that are volatile,flammable, and toxic.

[0111] In some embodiments, components of the gel forming reaction reactspontaneously at room temperature to form the macroporous gel. In otherembodiments, the gel forming reaction must be initiated. The gel formingreaction can be initiated by any known method, for example throughthermal activation or U.V. irradiation. The reaction is more preferablyinitiated by U.V. irradiation in the presence of a photoinitiator, asthis method has been found to produce larger macropores in the gel, andit accelerates the gel forming reaction more than the thermal activationmethod. Many suitable photoinitiators can be used, of which2-hydroxy-1[4-2(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959*), and 2,2-dimethoxy-2-phenylacetophenone (DMPA) are preferred.Other suitable photoinitiators include benzophenone, benzoin and benzoinethers such as benzoin ethyl ether and benzoin methyl ether,dialkoxyacetophenones, hydroxyalkylphenones, α-hydroxymethyl benzoinsulfonic esters. Thermal activation requires the addition of a thermalinitiator. Suitable thermal initiators include1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88),azobis(isobutyronitrile) (AIBN), potassium persulfate, ammoniumpersulfate, and benzoyl peroxide.

[0112] If the reaction is to be initiated by U.V. irradiation, aphotoinititor is added to the reactants of the gel forming reaction, andthe support member containing the mixture of monomer, cross-linkingagent and photoinitiator is subjected to U.V. irradiation at wavelengthsof from 250 nm to 400 nm, for a period of a few seconds to a few hours.With certain photoinitiators, visible wavelength light may be used toinititate the polymerization. To permit the initiation, the supportmaterial must have a low absorbance at the wavelength used, to permittransmittance of the UV rays through the support. Preferably, thesupport and macroporous gel reagents are irradiated at 350 nm for a fewseconds to up to 2 hours.

[0113] Preferably, thermally initiated polymerization is carried out at60-80° C. for a few minutes up to 16 hours.

[0114] The rate at which polymerization is carried out has an effect onthe size of the macropores obtained in the macroporous gel. As discussedearlier, when the concentration of cross-linker in a gel is increased tosufficient concentration, the constituents of the gel begin to aggregateto produce regions of high polymer density and regions with little or nopolymer, which latter regions are referred to as “macropores” in thepresent specification. It is this mechanism which is affected by therate of polymerization. When polymerization is carried out slowly, suchas when a low light intensity in the photopolymerization, theaggregation of the gel constituents has more time to take place, whichleads to larger pores in the gel. Alternatively, when the polymerizationis carried out at a high rate, such as when a high intensity lightsource is used, there is less time available for aggregation and smallerpores are produced.

[0115] Once the composite materials are prepared, they can be washedwith various solvents to remove any unreacted components and any polymeror oligomers that are not anchored within the support. Solvents suitablefor the washing of the composite material include water, acetone,methanol, ethanol, and DMF.

Uses of the Composite Material

[0116] The composite material of the invention can find use in manydifferent applications, where a liquid is passed through the macroporesof the gel. The liquid passed through the macropores can be selectedfrom, for example, a solution or a suspension, such as a suspension ofcells or a suspension of aggregates.

[0117] In some embodiments, the composite materials can be used toperform separations. Uses include size exclusion separations such asultrafiltration and microfiltration systems. The composite material ofthe invention is advantageous in these types of applications because ofthe wide range of pore-sizes available, and the ease with whichcomposite materials with different pore-sizes can be made. In someembodiments, it is preferred that the composite materials used forsize-exclusion separations are not fully occupied, i.e. that while allor substantially all the liquid that flows through the compositematerial flows through the macroporous gel, the void volume of thesupport member is not completely occupied by the macroporous gel.Composite materials having a non-fully occupied void volume where thedensity of the macroporous gel is greater at or adjacent to a firstmajor surface of the support member than the density at or adjacent to asecond major surface of the support member are referred to as beingasymmetric.

[0118] In those embodiments where the macroporous gel bears a charge,the combination of macropore surface charge and of controlled macroporesize produces a composite material that can be used in Donnan typeexclusion separations. In these instances composite materials areproduced that have high charge densities coupled with high hydraulicflows (flux). The high charge densities, coupled with high permeability,can be useful, for example, in adsorption of metal ions by givingcomposite materials with high ion-exchange capacities. The examples willdemonstrate that these composite materials can also be used for therecovery of proteins and other related molecules, and that the compositematerials of the invention exhibit high binding capacities.

[0119] Composite materials of the invention are also suitable for theseparation of biomolecules, such as proteins, from solution, as thebiomolecules may have specific interactions with ligands or bindingsites found in the macropores of the composite materials. The specificinteractions may involve electrostatic interactions, affinityinteractions or hydrophobic interactions. Examples of molecules or ions,including biological molecules or ions, that can be separated includeproteins such as albumins, e.g., bovine serum albumin, and lysozyme, butthey can also be used in the separation of supramolecular assembliessuch as viruses and cells. Examples of other biomolecules that can beseparated include γ-globulins of human and animal origins,immunoglobulins such as IgG, IgM, or IgE of both human and animalorigins, proteins of recombinant or natural origin including protein A,polypeptides of synthetic or natural origin, interleukin-2 and itsreceptor, enzymes such as phosphatase, dehydrogenase, etc., monoclonalantibodies, trypsin and its inhibitor, albumins of different origins,e.g., human serum albumin, chicken egg albumin, etc., cytochrome C,immunoglobulins, myoglobulin, recombinant human interleukin, recombinantfusion protein, nucleic acid derived products, DNA and RNA of eithersynthetic or natural origin, and natural products including smallmolecules. Biomolecule separations occur mostly in the macropores of thegel, but they can also take place, albeit at a slower rate, within thegel itself.

[0120] Some composite materials can be used as a reversible adsorbent.In these embodiments, a substance, for example a biomolecule, that isadsorbed in the macropores or in the mesh (micropore) of the gel can bereleased by changing the liquid that flows through the macroporous gel.Variations in the gel composition can be used to control the propertiesof the gel in terms of uptake and release of adsorbed substances.Another advantage of the composite material of the invention is that itcan be made in the form of a membrane, and membrane-based biomoleculerecovery is easier to scale up, less labor intensive, more rapid, andhas lower capital costs than the commonly used conventional packedcolumn chromatography techniques.

[0121] Some composite materials can also be used as solid supports forchemical synthesis or for cell growth. The reactants or nutrientsrequired for these processes can either continuously flow through themacropores of the composite material, or they can be left to resideinside the macropores and then pushed out at a later time. In oneembodiment, composite materials can be used in the stepwise productionof peptides. In such an application, amino acids are attached to themacropore surface, and amino acid solutions are sequentially passedthrough the macropores to prepare peptide chains. The formed peptide canthen be cleaved from the support by passing a suitable solvent throughthe macropores. Supports currently used to carry out this type ofsynthesis consist of beads with small pores that offer slow diffusioncharacteristics. The composite materials of the invention have a moreuniform pore structure, and they have through pores wherein the flow ofliquids is not controlled diffusion.

[0122] Application areas include but are not limited to pharmaceuticals,including biotechnology, food, beverage, fine chemicals, and therecovery of metal ions.

Uses of Responsive Composite Materials

[0123] The composite materials that comprise responsive macroporous gels(responsive composite materials) are preferably used to fractionatefluid components based on a size-exclusion mechanism. By this mechanism,steric hindrance is exerted on the convection and diffusion of amolecule or particle approaching the pores of a filtering device. Theimpediment to the transport is related to the ratio of the pore radiusin the filter to the radius of the molecule or particle. When this ratioapproaches to 1:1, molecules or particles will be completely retained bythe filtering device. By changing the pore radius, molecules orparticles of different sizes are permitted to pass through the pore andfractionation by size is achieved.

[0124] The responsive composite materials are well adapted to sizeexclusion mechanisms as the pores of these materials have a narrowpore-size distribution. This means that size exclusion separations canbe achieved for molecules that have molecular weights that are muchcloser than normally permitted by conventionalultrafiltration/microfiltration membranes. In some embodiments, the sizedifference between the molecules being separated by the responsivecomposite materials of the invention can be as low as about 0.9 nm. Inother embodiments, the resolution is as low as about 0.5 nm. While someof the known ultrafiltration membranes do have narrow pore-sizedistributions (e.g. track etched membranes), these membranes have thedisadvantage of being costly and of having relatively low fluxes.

[0125] In those embodiments where the composite materials arehydrophilic, the composite materials are well adapted to thesize-separation of proteins as there is little or no adsorption ofmolecules to the composite material. Normally, proteins are prone tonon-specific binding. Such responsive composite materials are thussuitable for the separation of a multi-component protein mixture intodiscrete fractions based only on the size of the protein fractions. Thisseparation of proteins through an exclusively size-exclusion methoddiffers from the methods known in the art for separating proteins, wheredifferences in size are used in conjunction with other physicochemicaleffects such as electrostatic charge, electrostatic double layer, andhydrophobic interaction effects, which physiochemical effects can leadto non-specific binding or even denaturation of the protein. Use of thecomposite material to separate proteins on a size-exclusion basis thusavoids the denaturation of the protein or its loss due to irreversiblebinding. The separation of proteins with the responsive compositematerial of the invention is a very gentle process that does not involvestrong interactions between the protein and the separation medium, whichpermits the recovery of proteins with higher overall efficiencies, amajor factor in the economics of therapeutic protein recovery.

[0126] One example of separations using the responsive compositematerials is the separation of human serum albumin (HSA) (size of 60kDa) from human immunoglobulin G (IgG) (size of 160 kDa). Size basedseparations of proteins are possible at fixed environmental conditions.With the responsive macroporous gel in a swollen or partially swollenstate, e.g., at a low salt concentration in the feed with gels havingweak acid or base functionalities, the protein with the lower molecularweight (e.g., human serum albumin) can be freely transmitted through thecomposite material, while the protein with the higher molecular weight(e.g., human immunoglobulin G) can be retained. When operated at a fixedenvironmental condition, the ultrafiltration process generates twoproduct streams, and the fixed environment mode is suitable forfractionating binary mixtures, i.e. one protein from another.

[0127] The responsive composite materials of the invention can also beused to generate more than two product streams through size-basedseparations utilising the dynamic pore-size capabilities of theresponsive composite material. This multi-component separation ispossible as the change in membrane pore-size in response to change inenvironmental condition is gradual. When operated in this mode (i.e.with change in environmental condition), the process is suitable forseparating proteins from a multi-protein mixture. In such a process, theenvironment is changed either in a stepwise fashion or gradually byappropriately altering the environment of the responsive gel. Forexample, changing the pH of the bulk medium used to carry out thefiltration process using binary or ternary buffer systems can bringabout a change in pore-size, and hence a sequential size basedseparation. When operated in the step change mode, each step willgenerate a fraction, each fraction containing proteins smaller than inthe next fraction. If n number of fractions are generated, (n−1) ofthese will be obtained in the permeate and the n^(th) fraction will bein the retentate.

[0128] The multi-component separations presented above represent acompletely new use of ultra- and nano-filtration membranes. This type ofmulti-component separation is also referred to as chromatographicfiltration. Some specific applications of this new type of separationinclude:

[0129] (a) fractionation of hen egg white components;

[0130] (i) LMW compounds such as Avidin (MW<1000);

[0131] (ii) Lysozyme (MW 14,100);

[0132] (iii) Ovalbumin (MW 47,000);

[0133] (iv) Conalbumin (MW 80,000);

[0134] (b) fractionation of human plasma proteins;

[0135] (i) Human serum albumin (HSA, MW 67,000);

[0136] (ii) Human immunoglobulin G (HIgG, MW 155,00);

[0137] (iv) Other human immunoglobulins (e.g. HIgM, MW>300,000);

[0138] (c) fractionation of dextran into molecular weight basedfractions;

[0139] (d) fractionation of PEG into molecular weight based fractions;

[0140] (e) fractionation of polymers into molecular weight basedfractions; and

[0141] (f) fractionation of micron-sized particles into size basedfractions.

[0142] While the composite materials of the present invention are quitesuitable for the separation of bulk materials, they can also be used toseparate components on a smaller volume scale. For example, theresponsive composite material can be used to separate biochemicalsubstances such as antibodies, other bioactive proteins, hormones,polysaccharides and nucleic acids, prior to analysis. Many of thecurrently used biospecific analytical methods, e.g. Enzyme Linked ImmunoSorbent Assay (ELISA) are based on the binding of the above substances,or the binding of substances which biospecifically interact with thesesubstances (e.g. antibodies, antigens, ligands, and substrateanalogues), onto solid surfaces such as polystyrene (as in ELISA) oronto synthetic membranes (as in immuno-blotting). The detection limitsof these tests are frequently limited by the surface area available indevices, such as microwell plates or blotted sections of flat sheetmembranes. Another limitation imposed by attachment of material ontosolid surfaces is the likelihood of steric hindrance affecting thebiospecific recognitions on which these tests are based. Biospecificanalytical methods which rely on solution phase recognition and bindingare also available, e.g. Radio Immuno Assay (RIA). These methodsfrequently rely on the use of porous synthetic membranes for retainingand enriching substances which are to be analyzed. However, the fixednature of the permeability of these membranes could prove to be alimiting factor. The use of responsive composite materials wouldfacilitate sequential removal of substance from solutions containingsubstances to be analyzed and thus facilitate analysis which would notbe feasible with fixed permeability membranes. The responsive membranescan also be utilised to facilitate removal from test solutions ofsubstances which are likely to interfere with the assays.

[0143] While the responsive composite materials are especially suitablefor use in size-exclusion separation, they can nonetheless be used inDonnan type separations and specific binding separations by theincorporation of appropriate monomers or polymers in the macroporousgel.

[0144] The responsiveness of composite material also permits the abilityto open the pores of a membrane (made from the composite material) afteruse, and to then return and readjust the pores to their initial valuesby reversing the environmental change. Opening of the pores facilitatesthe cleaning of these membranes by removing a fouling material, therebyprolonging the effective use of the membrane.

EXAMPLES

[0145] The following examples are provided to illustrate the invention.It will be understood, however, that the specific details given in eachexample have been selected for purpose of illustration and are not to beconstrued as limiting the scope of the invention. Generally, theexperiments were conducted under similar conditions unless noted.

EXPERIMENTAL Materials Used

[0146] The monomers used were acrylamide (AAM),2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS),(3-acrylamidopropane)trimethylammonium chloride (APTAC),diallyldimethylammonium chloride (DADMAC), ethylene glycoldimethacrylatecrylate (EDMA), glycidyl methacrylate (GMA),N,N′-methylenebisacrylamide (BIS), methacrylic acid (MAA), acrylic acid(AA), and trimethylolpropane triacrylate (TRIM). The polymers used werebranched poly(ethylene imine) (BPEI) of an average molecular weight (MW)of 25000 Da, poly(ethylene glycol) (PEG) of average molecular weight of200, 1000, 2000, 4000 and 10000 Da, and poly(allylammoniumhydrochloride) (PAH) of an average molecular weight of 60000 Da. Thecross-linker used for BPEI was ethylene glycol diglycidyl ether (EDGE).

[0147] The solvents used were cyclohexanol (CHX), methylene chloride(CH₂Cl₂), deionized water, 1,4-dioxane, N,N-dimethylformamide (DMF),dodecanol (DDC), glycerol, methanol, 1-octanol, and 1-propanol.

[0148] The free radical polymerization initiators used were2-hydroxy-1-[4-(2-hydoxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959), 2,2-dimethoxy-2-phenylacetophenone (DMPA), and1,1′-azobis(cyclohexanecarbonitrile) (VAZO® catalyst 88).

[0149] Proteins used were bovine serum albumin (BSA), lysozyme, humanserum albumin (HSA) and human immunoglobulin (HIgG). Other chemicalsused were acryloyl chloride, hydrochloric acid, sodium azide, sodiumchloride, sodium hydroxide, triethylamine,tris(hydroxymethyl)aminomethane (TRIS), 4-morpholineethanesulfonic acid(MES) and buffers (Tris Buffer).

[0150] The porous supports used were poly(propylene) thermally inducedphase separation (TIPS) membranes PP1545-4 with an average pore diameterof 0.45 μm, thickness of 125 μm, and porosity of 85 vol-%, produced by3M Company, and PP1183-3X of an average pore diameter of 0.9 μm,thickness of 87 μm, and porosity of 84 vol-%, both produced by 3MCompany, and non-woven meltblown poly(propylene) TR2611A of a mean poreflow diameter of 6.5 μm, thickness of 250 μm , and porosity of 89.5vol-% produced by Hollingworth & Vose Company.

Preparation of Composite Materials

[0151] The composite materials of the invention can be preparedaccording to the following general procedure. A weighed support memberwas placed on a poly(ethylene terephthalate) (PET) or poly(ethylene)(PE)sheet and a monomer or polymer solution was applied the sample. Thesample was subsequently covered with another PET or PE sheet and arubber roller was run over the sandwich to remove excess solution. Insitu gel formation in the sample was induced by polymerization initiatedby irradiation with the wavelength of 350 nm for the period of 10 to 120minutes or by heating the sandwich at 60-80° C. for 2 hours. Theirradiation was typically carried out using a system containing four 12″long lamps, approx. 1.5″ spaced and emitting light at 365 nm with theoutput energy of approx. 0.1 Watt/inch. The system was equipped with asmall fan to dissipate the heat (no other temperature control). Theirradiated sample was located at approx. 5″ distance from the lamps. Incase when preformed polymer and in situ cross-linking was used to formthe gel, the sandwich was left at room temperature until thecross-linking reaction was completed, typically for 2-16 hours. Theresulting composite material was thoroughly washed with a suitablesolvent or a sequence of solvents and stored in a 0.1 wt-% aqueoussolution of sodium azide to prevent bacterial growth. In order todetermine the amount of gel formed in the support, the sample was driedin vacuum at room temperature to a constant mass. The mass gain due togel incorporation was calculated as a ratio of an add on mass of the drygel to the initial mass of the porous support.

Flux Measurements

[0152] Water flux measurements through the composite materials werecarried out after the samples had been washed with water. As a standardprocedure, a sample in the form of a disk of diameter 7.8 cm was mountedon a sintered grid of 3-5 mm thickness and assembled into a cellsupplied with compressed nitrogen at a controlled pressure. The cell wasfilled with deionized water or another feed solution and a desiredpressure was applied. The water that passed through the compositematerial in a specified time was collected in a pre-weighed containerand weighed. All experiments were carried out at room temperature and atatmospheric pressure at the permeate outlet. Each measurement wasrepeated three or more times to achieve a reproducibility of ±5%.

[0153] The water flux, Q_(H2O) (kg/m²h), was calculated from thefollowing relationship:$Q_{H_{2}O} = \frac{\left( {m_{1} - m_{2}} \right)}{A \cdot t}$

[0154] where m₁ is the mass of container with the water sample, m₂ isthe mass of container; A is the active membrane surface area (38.5 cm²)and t is the time.

[0155] The composite material of the invention may have water fluxvalues that are smaller than those of the unfilled support member, withpossible flux reduction of about a factor of two to about of a factor ofa few hundred depending on the application. For ultrafiltrationapplication, the flux may be reduced by a factor of about ten to about afew hundred.

[0156] The hydrodynamic Darcy permeability, k (m²) of the membrane wascalculated from the following equation$k = \frac{Q_{H_{2}O}{\eta\delta}}{3600\quad d_{H_{2}O}\Delta \quad P}$

[0157] where η is the water viscosity (Pa·s), δ is the membranethickness (m), d_(H2O) is the water density (kg/m³), and ΔP (Pa) is thepressure difference at which the flux, Q_(H2O), was measured.

[0158] The hydrodynamic Darcy permeability of the membrane was used toestimate an average hydrodynamic radius of the pores in the porous gel.The hydrodynamic radius, r_(h), is defined as the ratio of the porevolume to the pore wetted surface area and can be obtained from theCarman-Kozeny equation given in the book by J. Happel and H. Brenner,Low Reynolds Number Hydrodynamics, Noordhof Int. Publ., Leyden, 1973, p.393: $k = \frac{ɛ\quad r_{h}^{2}}{K}$

[0159] where K is the Kozeny constant and ∈ is the membrane porosity.The Kozeny constant K≈5 for porosity 0.5<∈<0.7. The porosity of themembrane was estimated from porosity of the support by subtracting thevolume of the gel polymer.

Protein Adsorption/Desorption Experiment

[0160] Protein adsorption experiments were carried out with twoproteins, namely, bovine serum albumin (BSA) and lysozyme. In the caseof experiments with a positively charged composite material in the formof a membrane, the membrane sample was first washed with distilled waterand subsequently with a TRIS-buffer solution (pH=7.8). In an adsorptionstep, a composite material sample in a form of a single membrane disk ofdiameter 7.8 cm was mounted on a sintered grid of 3-5 mm thickness in acell used for water flux measurements and described above. A BSAsolution, comprising from 0.4 to 0.5 mg BSA per ml of buffer solution,was poured to the cell to give a 5 cm head over the composite material.This hydrostatic pressure of 5 cm was kept constant by further additionsof the BSA solution. In a modification of this method, the cell waspressurised with compressed nitrogen. The flow rate was measured byweighing the amount of permeate as a function of time. Typical valuesvaried between 1 and 5 ml/min. Permeate samples were collected at 2-5min intervals and analyzed by UV analysis at 280 nm. Following theadsorption step, the composite material in the cell was washed withabout 200 ml of the TRIS-buffer solution, and desorption was carried outwith a TRIS-buffer solution containing 1M NaCl at 5 cm head pressure orunder a controlled pressure of compressed nitrogen. The permeate sampleswere collected at 2-5 min intervals and tested by UV analysis at 280 nmfor BSA content.

[0161] For negatively charged composite materials, a solution oflysozyme in a MES buffer solution having a pH of 5.5 and a lysozymeconcentration of 0.5 g/L was used in a procedure similar to thatdescribed above for BSA and positively charged materials. The flow rateduring the protein adsorption was again kept within 1-5 ml/min. Prior tothe desorption of the protein, the membrane was washed by passing with200 ml of the buffer solution. The desorption of the protein was carriedout using a MES buffer solution (pH=5.5) containing 1M NaCl in the sameway as described above for the desorption of BSA. The lysozyme contentin the collected samples was determined by UV spectrophotometry at 280nm.

[0162] In other examples, protein adsorption tests involve stacks ofseveral membranes of diameter of 19 mm mounted into a Mustang® CoinDevice manufactured by Pall Corporation and the protein solution wasdelivered to the membrane stack at controlled flow rate using aperistaltic pump. The permeate fractions were collected and analyzed inthe same way as described above. The desorption of the proteins wascarried in a similar way as described above, with buffered 1M NaCldelivered to the membrane stack by using the pump instead of gravity orcompressed nitrogen pressure.

Protein Separation Experiment

[0163] The experimental method used to examine the separation propertiesof the responsive composite materials of this invention inprotein-protein fractionation processes is based on the pulsed injectionultrafiltration technique and its derivatives developed by Ghosh and hisco-workers and described in the following articles: R.Ghosh and Z. F.Cui, Analysis of protein transport and polarization through membranesusing pulsed sample injection technique, Journal of Membrane Science,vol. 175, no. 1 (2000) p. 75-84; R.Ghosh, Fractionation of biologicalmacromolecules using carrier phase ultrafiltration, Biotechnology andBioengineering, vol. 74, no. 1 (2001) p. 1-11; and R. Ghosh, Y. Wan, Z.F. Cui and G. Hale, Parameter scanning ultrafiltration: rapidoptimisation of protein separation, Biotechnology and Bioengineering,vol. 81 (2003) p. 673-682 and incorporated herein by reference. Theexperimental set-up used was similar to that used for parameter scanningultrafiltration as described in article by R. Ghosh, Y. Wan, Z. F. Cuiand G. Hale, Parameter scanning ultrafiltration: rapid optimisation ofprotein separation, Biotechnology and Bioengineering, vol. 81 (2003) p.673-682.

[0164] A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the responsive compositematerial experiments was one with a low salt concentration (typically5-10 mM NaCl). In all these experiments the carried phase was switchedto one with a high salt concentration (typically 1 M NaCl). The changein salt concentration within the membrane module could be tracked byobserving the conductivity of the permeate stream. The change intransmembrane pressure gave an idea about the change in membranehydraulic permeability with change in salt concentration.

Example 1

[0165] This example illustrates the formation of an unsupported porousgel, which can be used as the macroporous gel to prepare the compositematerial of the invention.

[0166] A solution containing 3.33 g of(3-acrylamidopropane)trimethylammonium chloride (APTAC) monomer as a 75%aqueous solution, 0.373 g of N,N′-methylenebisacrylamide (BIS)cross-linker, and 0.0325 g of Irgacure® 2959 photoinitiator dissolved in25 ml of a dioxane:dimethylformamide:water mixture, with the solventvolume ratio of 71:12:17, respectively, was prepared. In this solventmixture, dioxane is a poor solvent while DMF and water are goodsolvents. A total monomer concentration (APTAC and BIS) of 0.58 mol/Lwas thus obtained. The cross-linking degree was 20 mol %, based onAPTAC. 5 ml of this solution was placed in a glass vial and subjected toUV irradiation at 350 nm for 2 hrs. A white gel was formed which waswashed thoroughly with de-ionized water to exchange the reaction solventand remove the unreacted monomer or soluble oligomers.

[0167] The gel formed was mechanically very weak. A sample of the gelwas examined using an environmental scanning electron microscope (ESEM)with water vapor present in the sample chamber to prevent drying of thegel. The micrograph, shown in FIG. 1, has dark, cavernous areas thatindicate that a macroporous gel was formed.

Example 2

[0168] This example illustrates a method of preparing a positivelycharged composite material of the present invention using the monomersolution of composition described in example 1 applied to a sample ofthe poly(propylene) porous support PP1545-4. The composite material wasprepared according to the general procedure described above using UVirradiation at 350 nm for 2 hours. After polymerization, the compositematerial was washed with with de-ionized water for 48 hrs.

[0169] Mass gain of the resulting composite material after drying was107 wt %, water flux was 1643±5 kg/m²h at 50 kPa and Darcy permeabilitywas 9.53×10⁻¹⁶ m².

[0170] The morphology of the gel-incorporated composite material wasexamined using ESEM in the same manner as described in Example 1. TheESEM micrograph shown in FIG. 2 shows that the macroporous gel has beenincorporated into the host membrane. The micrograph shows a similarstructure to that of the unsupported macroporous gel shown in FIG. 1 andlittle evidence of the microporous support member.

Example 3

[0171] This example illustrates a method of preparing a negativelycharged composite material of the present invention, with a weak acidfunctionality.

[0172] 5.50 g of vacuum-distilled methacrylic acid (MAA) monomer, 0.4925g of N,N′-methylenebisacrylamide cross-linker and 0.1503 g of Irgacure®2959 photoinitiator were dissolved in 25 ml of a dioxane:DMF solventmixture with a volume ratio of 9:1, respectively, to prepare thestarting monomer solution. The composite material was prepared using thepoly(propylene) PP1545-4 support and the general procedure for thephotoinitiated polymerization described above. The irradiation time usedwas 2 hours and the resulting membrane was washed with DMF for 24 hrsfollowed by a 48 hr wash with deionized water. The mass gain of theresulting dried membrane was 231 wt %, water flux was 4276±40 kg/m²h at50 kPa and Darcy permeability was 2.64×10⁻¹⁵ m².

[0173] The protein (lysozyme) absorption/desorption characteristics ofthe composite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 3. It can be seen that even with the singlemembrane disk, a relatively steep break through curve is obtainedindicating a uniform and narrow pore size distribution in the membrane.The composite material has a breakthrough lysozyme binding capacity of42.8 mg/mL. A desorption experiment with a buffer solution containing 1MNaCl indicated that the recovery of protein was 83.4%.

Example 4

[0174] This example illustrates the effect of the total monomerconcentration and solvent mixture on the hydraulic flow rate (flux) ofcomposite membranes with weak acid functionality of the type describedin Example 3.

[0175] A series of composite membranes (MAA1 through MAA5) were preparedusing monomer solutions of chemical compositions listed in Table 1 andthe porous support PP1545-4. The preparation procedure described inExample 3 was employed. TABLE 1 The effect of the total monomerconcentration and solvent mixture on water flux of composite membranesTotal Monomer Solvent Concentration Cross-linking Mixture Mass Flux at(MAA + BIS) Degree (volume part) Gain 50 kPa Sample I.D. (mol/L) (mol-%)Dioxane DMF (wt %) (kg/m² · h) MAA1 1.71 5 8 2  71 12.2 ± 0.1  MAA2 2.195 8 2 153 94 ± 14 MAA3 2.68 5 8 2 177 1265 ± 111  MAA4 3.66 5 8 2 3001800 ± 9   MAA5 2.68 5 9 1 231 4276 ± 40 

[0176] As can be seen from Table 1, the hydraulic flow rate (flux) ofcomposite membranes of the present invention can be tuned by adjustingthe monomer loading in the solution. Contrary to the typical trendsfound with homogeneous gels, for which an increase in gel density isfollowed by decrease in permeability, the increase in the mass gain inthe membranes of this series results in the flux increase. Furtherincrease in flux is achieved when the concentration of the poor solvent(dioxane) in the solvent mixture is increased (compare samples MAA3 andMAA5).

Example 5

[0177] This example illustrates a method of preparing a negativelycharged composite material of the present invention that has strong acidfunctionality.

[0178] A solution containing 2.50 g2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monomer, 0.372 gN,N′-methylenebisacrylamide cross-linker and 0.0353 g Irgacure® 2959photo-initiator, dissolved in 25 ml of a dioxane:H₂O mixture with avolume ratio 9:1, respectively, was used. A composite material wasprepared from the solution and the support PP1545-4 using thephotoinitiated polymerization according to the general proceduredescribe above. The irradiation time used was 1 hour at 350 nm. Afterpolymerization, the membrane was extracted with de-ionized water for 48hrs. The mass gain of the resulting membrane was 74.0 wt %, water fluxwas 2559±40 kg/m²h at 50 kPa and Darcy permeability was 1.58×10⁻¹⁵ m².

Example 6

[0179] This example illustrates further the effect of the solventmixture composition and the cross-linking degree on the hydraulic flowrate of composite membranes with the strong acid functionality. A seriesof composite membranes (AMPS1 through AMPS5) was prepared using chemicalcompositions listed in Table 2 following the general preparationprocedure and the irradiation conditions as in example 5. TABLE 2 Theeffect of solvent mixture on water flux of the composite membranes TotalCross- Monomer linking Solvent mixture Mass Flux at Sample Conc. Degree(volume part) gain 50 kPa I.D. (mol/L) (mol %) Dioxane DMF H₂O (wt %)(kg/m²h) AMPS1 0.48 20 5 5 0  92 3.2 ± 0.0 AMPS2 0.48 20 8 2 0 100 575 ±12  AMPS3* 0.48 20 9 0 1  74 2559 ± 9   AMPS4 0.48 10 8 2 0 100 8.4 ±0.0

[0180] As can be seen, a similar pattern to that described in example 4was observed with regards to the relationship between solubility ofpolymer in the solvent and water flux of composite membranes. Comparisonof AMPS2 with AMPS 4 shows that hydraulic flow rate (flux) of acomposite membrane can also be adjusted by the degree of cross-linking.

Example 7

[0181] This example illustrates the effect of introducing a neutralco-monomer into a negatively charged composite material of the presentinvention.

[0182] A solution containing 1.750 g of2-acrylamido-2-methyl-1-propanesulfonic acid, 0.485 g of acrylamide,0.868 g of N,N′-methylenebisacrylamide cross-linker, and 0.044 g ofIrgacure® 2959 photo-initiator, dissolved in 25 ml of a dioxane:DMF:H₂Omixture with a volume ratio 8:1:1, respectively, was prepared. Acomposite material was prepared from the solution and the supportPP1545-4 using the photoinitiated polymerization according to thegeneral procedure describe above. The irradiation time used was 1 hourat 350 nm. After polymerization, the membrane was extracted withde-ionized water for 48 hrs.

[0183] The mass gain of the resulting membrane was 103 wt %, water fluxwas 7132±73 kg/m².h at 100 kPa, and Darcy permeability was 4.40×10⁻¹⁵m².

Example 8

[0184] This example illustrates one method of making a positivelycharged composite material of this invention.

[0185] A 15 wt-% solution was prepared by dissolvingdiallyldimethylammonium chloride (DADMAC) monomer andN,N′-methylenebisacrylamide (BIS) cross-linker in a molar ratio of 5:1,respectively, in a solvent mixture containing 37 wt-% water, 45 wt-%dioxane and 18 wt-% DMF. The photo-initiator Irgacure® 2959 was added inthe amount of 1% with respect to the mass of the monomers.

[0186] A composite material was prepared from the solution and thesupport PP1545-4 using the photoinitiated polymerization according tothe general procedure describe above. The irradiation time used was 30minutes at 350 nm. The composite material was removed from between thepolyethylene sheets, washed with water and TRIS-buffer solution andstored in water for 24 hrs.

[0187] Several samples similar to that described above were prepared andaveraged to estimate the mass gain of the composite material. Thesubstrate gained 42.2% of the original weight in this treatment.

[0188] The composite material produced by this method had a water fluxin the range of 2100-2300 kg/m² hr at 70 kPa and Darcy permeability of9.87×10⁻¹⁶ m².

[0189] The protein (BSA) adsorption characteristic of the compositematerial was examined using the general procedure for a single membranedisk described above. The concentration of the protein used in thisexperiment was 0.4 g/L in 50 mM TRIS-buffer. The flow rate was 2-4ml/min. A plot of the concentration of BSA in the permeate vs. thepermeate volume is shown in FIG. 4. The composite material had a BSAbinding capacity of 48-51 mg/ml. The BSA desorption was found to be inthe range of 78-85%.

Example 9

[0190] This example illustrates that by adding a neutral monomer to thecharged monomer used in Example 6 the protein binding capacity can besubstantially increased.

[0191] A 10 wt-% solution was prepared by dissolvingdiallyldimethylammonium chloride (DADMAC) and acrylamide (AAM), in theratio 80:20, in a solvent mixture containing 63 wt-% dioxane, 18 wt-%water, 15 wt-% DMF, and 4 wt-% dodecanol. N,N′-methylenebisacrylamidecross-linker was added to the monomer solution to obtain 40% (mol/mol)cross-linking degree. The photoinitiator Irgacure® 2959 was added in theamount of 1% with respect to the total mass of monomers.

[0192] A composite material was prepared from the solution and thesupport PP1545-4 using the photoinitiated polymerization according tothe general procedure describe above. The irradiation time used was 20minutes at 350 nm. The composite material was removed from between thepolyethylene sheets, washed with water, TRIS-buffer solution and storedin water for 24 hrs.

[0193] A similar sample to that described above was prepared and used toestimate the mass gain of the composite material. The substrate gained80% of the original weight in this treatment.

[0194] The composite material produced by this method had a water fluxin the range of 250 kg/m² hr at 70 kPa and Darcy permeability was1.09×10⁻¹⁶ m².

[0195] The protein (BSA) adsorption characteristic of the compositematerial was examined using the general procedure for a single membranedisk described above. The protein concentration was 0.4 g/L in a 50 mMTRIS buffer solution. The flow rate of absorption experiment wasadjusted to 2 4 ml/min. The composite material had a BSA bindingcapacity of 104 mg/ml.

Example 10

[0196] This example illustrates the formation of a supported porous gelcomposite material by cross-linking of a pre-formed polymer.

[0197] Three separate solutions were prepared with the followingcompositions: (A) 20 g of branched poly(ethyleneimine) (BPEI) (25,000Da) in 50 ml of methanol, (B) 20 g of poly(ethyleneglycol) PEG (˜10,000Da) in 50 ml of methanol, and (C) ethyleneglycol diglycidyl ether (0.324g) in 5 ml of methanol.

[0198] A mixture of the three solutions was prepared consisting of 2 mlof (A), 3 ml of (B), and 5 ml of (C). A portion of this resultingsolution was allowed to stand in a vial overnight when a phaseseparation was observed. Examination of the morphology of the upperclear gel layer indicated that it was macroporous.

[0199] The same mixed solution was spread on a sample of poly(propylene)support PP1545-4 using the techniques described in the generalprocedure. The membrane was sandwiched between twopoly(ethyleneterephthalate) sheets and allowed to stand overnight. Thecomposite material was extracted with methanol at room temperature for24 h, and a mass gain of 95% was observed. The water flux of thecomposite material was 6194 kg/m² h at 100 kPa and Darcy permeabilitywas 4.89×10⁻¹⁰ m².

[0200] The dynamic protein absorption capacity of the composite materialwas measured using a BSA solution (0.4 mg/mL) in the method for a singlemembrane disk described in the general section above. It had a capacityof 68 mg/ml before breakthrough.

Example 11

[0201] This example illustrates the effect of monomer mixturecomposition and the polymerization conditions on the hydraulicproperties of composite materials prepared by in situ polymerization ofglycidyl methacrylate (GMA) with ethylene dimethacrylate (EDMA) used asa cross-linker. The solvents used were dodecanol (DDC), cyclohexanol(CHX), and methanol. A porous polypropylene support membrane PP1545-4and two modes of initiation of in situ polymerization were usedaccording to the general procedure described above. In thephotopolymerization mode, 2,2-dimethoxy-2-phenylacetophenone (DMPA) wasused as a photoinitiator while the thermal polymerization was initiatedby 1,1′-azobis(cyclohexanecarbonitrile). In both modes, thepolymerization was carried out for 2 hours.

[0202] The polymerization conditions and properties of the compositematerials containing porous poly(glycidyl methacrylate-co-ethylenediacrylate) are presented in Table 3. TABLE 3 Porous poly(glycidylmethacrylate-co-ethylene diacrylate)-filled composite materials TotalMonomer Initiation Mode Mass Darcy Hydrodynamic Membrane Concentrationof Gain Permeability radius ID wt-% Solvent Polymerization wt-% m² nmAM612 43.8 DDC/CHX 9/91 Photo 276.8 6.96 × 10⁻¹⁶ 95.1 AM614 22.9 DDC/CHX9/91 Photo 144.3 2.77 × 10⁻¹⁵ 148.5 AM615 47.6 DDC/CHX 9/91 Thermal237.0 1.66 × 10⁻¹⁷ 13.0 AM616 24.9 DDC/CHX 9/91 Thermal 157.5 9.15 ×10⁻¹⁶ 86.4 AM619 48.6 Methanol Photo 265.0 2.48 × 10⁻¹⁵ 163.8

[0203] The mass gain obtained in this series of composite materials isproportional to the total monomer concentration in the polymerizationmixture. Membranes AM612, AM615, and AM619 were prepared using highconcentration of monomers while in membranes AM614 and AM616 the monomerconcentration was cut approximately by half (Table 3).

[0204] The high values of the pure water flux measured at 100 kPa oftransmembrane pressure (Table 3) indicate that the pore-filling materialis macroporous. The pure water flux and, consequently, the hydraulicradius are affected not only by the mass gain but also by thepolymerization mode. As shown in FIG. 5, the hydraulic radius is alinear function of the mass gain with the slope depending on thepolymerization mode. The absolute value of the negative slope in thethermal polymerization is twice that of the photopolymerized compositematerials. This means that photopolymerized composite materials havelarger pores than that of the thermally polymerized ones at the samemass gains. Thus, the photo-initiated polymerization, which is fasterthan the thermally-initiated one, produces larger pores. Since themonomer conversion is practically the same in both cases ofpolymerization (similar mass gains), the presence of the poly(propylene)substrate either through its hydrophobic nature or by creatingmicroscopic confinements for the polymerization affects the poreformation and the final structure of the pore-filling materials.

[0205] By changing the solvents from dodecanol/cyclohexanol 9/91 tomethanol, which is cheaper and environmentally more acceptable than theother solvents, a composite material with very high flux was obtained(membrane AM619, Table 3). The composite material was produced from theconcentrated monomer mixture and had flux comparable with that of themembrane AM614 which had a mass gain almost twice as low as that ofAM619.

[0206] This and subsequent examples illustrate a feature of somecomposite materials of the invention. With the capability to change thecomposition and concentration of the monomers and solvents, there can beproduced stable composite materials with different porous structures. Asshown in Table 3, composite materials with larger pores can be made inthis way.

Example 12

[0207] This example illustrates further the effect of monomer mixturecomposition on the hydraulic properties of composite materials of thisinvention.

[0208] A series of composite materials have been prepared according tothe general procedure described above and containing porouspoly(acrylamide) gels formed by in situ photoinitiated polymerization ofacrylamide (AAM) and N,N′-methylenebisacrylamide (BIS) as a cross-linkerin the pores of a poly(propylene) support membrane. The porous supportmember used was poly(propylene) TIPS membrane PP1545-4.2,2-Dimethoxy-2-phenylacetophenone (DMPA) or1-[4-(2-hydroxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one(Irgacure® 2959) were used as photoinitiators. Irradiation was carriedout at 350 nm for 2 hours. Composition of the pore-filling solutions andthe properties of the resulting composite materials are summarized inTable 4. TABLE 4 Composition and properties of poly(acrylamide)-filledcomposite materials Total Degree Monomer Solvent 1 Solvent 2 DarcyHydrodynamic Membrane of XL Conc. Conc. Conc. Mass Permeability RadiusI.D. Wt-% wt-% Name wt-% Name wt-% Gain % m² nm AM606 18.0 13.3 Water86.6 None 0.0 111.7 9.3 × 10⁻¹⁸ 8.0 AM607 18.0 13.6 Water 67.9 Methanol18.4 107.8 2.5 × 10⁻¹⁸ 4.1 AM608 18.0 14.4 Water 71.8 Glycerol 13.7110.4 2.1 × 10⁻¹⁸ 3.8 AM609 16.8 12.0 Water 51.9 Glycerol 36.0 103.3 1.6× 10⁻¹⁸ 3.3 AM610 31.8 34.5 DMF 49.1 1-Propanol 16.4 307.2 3.9 × 10⁻¹⁷20.0 AM611 32.0 18.7 DMF 60.9 1-Propanol 20.5 130.3 5.3 × 10⁻¹⁶ 62.7AM617 32.2 34.9 DMF 48.4 1-Octanol 16.7 273.3 8.6 × 10⁻¹⁷ 29.0

[0209] Membranes AM606 through AM609 have been prepared using verysimilar concentration of monomers (12.0-14.4 wt-%) and a similar,relatively high, degree of cross-linking (16.8-18.0 wt-% of monomers).The mass gains obtained with these composite materials are also verysimilar. As shown in FIG. 6, there is a linear relationship betweentotal monomer concentration in the pore-filling solution and the massgain achieved after photopolymerization.

[0210] The high degree of cross-linking in the composite materialprepared from aqueous solution without non-solvent (AM606) leads torelatively high permeability. Surprisingly, the addition of methanol orglycerol, which are poor solvents for linear poly(acrylamide), to water,which is a good solvent for the linear polymer, brings about asubstantial reduction in the Darcy permeability and the hydrodynamicradius calculated on its basis. The reduction in permeability is higherwith glycerol than with methanol and increases with the amount ofglycerol in the solution.

[0211] The use of mixtures of poor solvents, such asN,N′-dimethylformamide and 1-propanol or 1-octanol, as well as thefurther increase of the degree of cross-linking and total monomerconcentration have been tested in membranes AM610, AM611, and AM617. Asshown in Table 4, substantially higher permeabilities and hydraulicradii are obtained with all these composite materials as compared to thecomposite materials prepared with water as one of the solvents. Thisoccurred despite an increase of the total monomer concentration; morethan double in membranes AM610 and AM617 than that in the membranesprepared with water as one of the solvents. Changing the other solventfrom 1-propanol in AM610 to 1-octanol in AM617 also brings aboutsubstantial increase in permeability and hydraulic radius.

[0212] Microscopic images of the surface of membrane AM610 are shown inFIGS. 7 (AFM) and 8 (ESEM). For comparison, an ESEM image of the nascentporous support member is also shown in FIG. 8. Both sets of images showa porous phase-separated gel covering the member surface with nodiscernible elements of the support member.

[0213] Membrane AM611 was prepared with DMF and 1-propanol but the totalmonomer concentration was just over half that of AM610. Membrane AM611shows very high flux and the hydraulic radius three times that of AM610.The ESEM images of the surface of this membrane are presented in FIG. 9.It shows a highly porous gel structure (top image) that resembles thebulk gel formed in some spots on the membrane surface but detached fromthe membrane (bottom picture).

[0214] A comparison of surfaces of membranes AM610 and AM611 ispresented in FIG. 10. The large difference in the size of the structuralelements in these two gels is clearly visible.

[0215] The composite materials prepared in this example can serve asultrafiltration membranes. It has been shown that the pore size of thecomposite material and, therefore, its separation properties, can becontrolled to achieve a wide range of values.

Example 13

[0216] This example illustrates the effect of pore size of the supportmember on the hydraulic flow rate (flux) through composite materials ofthis invention.

[0217] Two polypropylene support membranes of pore size 0.45 μm and 0.9μm, PP1545-4 and PP1183-3X, respectively, were used to produce compositematerials with the same monomer mixture containing 39.4 wt-% of glycidylmethacrylate and 9.2 wt-% of ethylene diacrylate in methanol, thushaving 48.6 wt-% of monomers and 18.9 wt-% of ethylene diacrylate(cross-linker) in the monomer mixture. The photoinitiator used was DMPAin the amount of 1.3 wt-% of monomers.

[0218] The composite materials were prepared according to the generalprocedure described above. The irradiation time was 2 hours at 350 nm.The resulting composite materials were washed with methanol followed bydeionized water. The composite materials were tested for water flux at100 kPa to calculate the Darcy permeability and hydraulic radius. Theresults are presented in Table 5. TABLE 5 Hydraulic properties ofcomposite membranes produced with substrates of different pore sizesAverage Standard Support pore Mass Gain Flux at 100 Hydrodynamichydrodynamic Deviation Membrane ID size (μm) (wt-%) kPa (kg/m²h) radius(nm) radius (nm) (%) AM619 0.45 265.0 8080.9 163.8 156.2 6.9 AM620 0.90296.8 7310.1 148.5

[0219] The data show that the hydraulic radius in both compositematerials is the same within an experimental error, proving that thecomposite materials contain macroporous gels of similar structure.

Example 14

[0220] This Example illustrates the synthesis of poly(ethylene glycol)(PEG, MW's 4000, 2000, 1,000, and 200) diacrylates, which can be used ascross-linkers to prepare the composite material of the invention.

[0221] The synthesis procedure used follows that described by N. Ch.Padmavathi, P. R. Chatterji, Macromolecules, 1996, 29, 1976, which isincorporated herein by reference. 40 g of PEG 4000 was dissolved in 150ml of CH₂Cl₂ in a 250-ml round bottom flask. 2.02 g of triethylamine and3.64 g of acryloyl chloride were added dropwise to the flask separately.Initially the reaction temperature was controlled at 0° C. with an icebath for 3 hrs, and then the reaction was allowed to warm to roomtemperature and kept for 12 hrs. The reaction mixture was filtered toremove the precipitated triethylamine hydrochloride salt. The filtratethen was poured into an excess of n-hexane. The colorless product,referred to as PEG 4000 diacrylate, was obtained by filtration anddrying at room temperature.

[0222] The same procedure was used with the PEG's of other molecularweights. The molar ratios of the PEG to acryloyl chloride were kept thesame as used above with PEG 4000.

Example 15

[0223] This example illustrates a further method of preparing anegatively charged composite material that has a high adsorptioncapacity for lysozyme.

[0224] A solution containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and 0.4 g ofacrylamide (AAM) as monomers, 0.25 g of N,N′-methylenebisacrylamide(BIS) and 1.0 g of PEG 4000 diacrylate obtained in Example 14 ascross-linkers, and 0.01 g of Iragure® 2959 as a photoinitiator wasprepared in 10 ml of solvent consisting of a 80:10:10 volume ratio ofdioxane, dimethylformamide (DMF), and water.

[0225] A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the composite material was prepared according tothe general procedure, with the irradiation carried out at 350 nm for 20minutes. After polymerization, the composite material was washedthoroughly with de-ionized water for 24 hrs.

[0226] Mass gain of the resulting composite material after drying was113.2 wt %, water flux was 366±22 kg/m²h at 100 kPa, and Darcypermeability was 2.26×10⁻¹⁶.

[0227] The protein (lysozyme) absorption/desorption characteristics ofthe composite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in the permeate versus the volumeof permeate is shown in FIG. 11. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a Lysozymebinding capacity of 103.9 mg/ml. A desorption experiment indicated thatthe recovery of protein was 64.0%.

Example 16

[0228] This example illustrates preparation of a negatively chargedcomposite material with the same nominal polymer compositon as inExample 15 but with much higher hydraulic flows (flux) and good lysozymeuptake capacity.

[0229] The monomer solution was produced by dilution of the solutionformulated in Example 15 with acetone with the mass ratio of 1:1.

[0230] A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the composite material was prepared according tothe general procedure. The irradiation time used was 2 hours. Afterpolymerization, the composite material was washed thoroughly withde-ionized water for 24 hrs.

[0231] The mass gain of the resulting composite material after dryingwas 51.1 wt % and water flux was 6039±111 kg/m².h at 100 kPa givingDarcy permeability of 3.73×10⁻¹⁵.

[0232] The protein (lysozyme) absorption/desorption characteristics ofthe composite material were examined using the general procedure for asingle membrane disk outlined earlier. The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min. Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 12. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a lysozymebinding capacity of 75.4 mg/ml. A desorption experiment indicated thatthe recovery of protein was 65.0%.

Example 17

[0233] This example illustrates a further preparation of a negativelycharged composite material that has a very high flux but lower proteinbinding capacity.

[0234] The monomer solution was produced by dilution of the solutionformulated in Example 15 with acetone with the mass ratio of 1:2.

[0235] A porous poly(propylene) support member in the form of a membrane(PP1545-4) was used and the preparation of composite material wascarried out according to the general procedure described above. UVinitiated polymerization was carried out for 2 hours. Afterpolymerization, the composite material was washed thoroughly withde-ionized water for 24 hrs.

[0236] The mass gain of the resulting composite material after dryingwas 34.4 wt % and water flux was 12184±305 kg/m²h at 100 kPa givingDarcy permeability of 7.52×10⁻¹⁵.

[0237] The protein (lysozyme) absorption/desorption characteristics ofthe composite material were examined using the general procedure for asingle membrane disk outlined earlier. (The concentration of the proteinused in this experiment was 0.5 g/L in a 10 mM MES buffer at pH 5.5. Theflow rate of adsorption experiment was regulated to be 2-4 ml/min.) Aplot of the concentration of lysozyme in permeate versus the volume ofpermeate is shown in FIG. 13. It can be seen that a relatively steepbreak through curve is obtained. The composite material had a lysozymebinding capacity of 53.5 mg/ml. A desorption experiment indicated thatthe recovery of protein was 99.0%.

[0238] Examples 15, 16 and 17 show that it is possible to control theloading of porous gel into the host membrane thereby controlling thewater flux at a defined pressure (100 kPa in the data given in theexamples) and also that the lysozyme uptake is related to the mass ofincorporated porous gel.

Example 18

[0239] This example illustrates preparation of a negatively chargedmembrane that has both good protein adsorption capacity and good fluxusing a macromonomer.

[0240] A monomer solution containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.25 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG 2000 macromonomer obtained in Example 14,dissolved in 10 ml of a dioxane-(DMF)-water mixture with a volume ratio80:10:10, respectively, was prepared.

[0241] A microporous poly(propylene) support member in the form of amembrane, support PP1545-4, was used together with the general proceduredecribed above. The irradiation time used was 20 minutes. Afterpolymerization, the membrane was washed thoroughly with de-ionized waterfor 24 hrs.

[0242] The mass gain of the resulting membrane after drying was 108.4 wt% and water flux was 1048±4 kg/m²h at 100 kPa giving Darcy permeabilityof 6.47×10⁻¹⁶.

[0243] The protein (lysozyme) adsorption/desorption characteristics ofthe membrane were examined using the general procedure for a singlemembrane disk outlined earlier. A relatively steep break through curvewas obtained. The membrane had a lysozyme binding capacity of 88.7mg/ml. The desorption experiment indicated that the recovery of proteinwas 64.0%.

Example 19

[0244] This example in combination with example 18 above furtherillustrates that the protein binding capacity and flow characteristicsof a membrane can be tuned.

[0245] The monomer solution was produced by dilution of the solutionformulated in Example 18 with acetone with the mass ratio of 1:1.

[0246] A porous poly(propylene) support member in the form of amembrane, support PP1545-4, was used along with the general procedurefor the preparation of composite materials described above. Theirradiation time used was 90 minutes. After polymerization, the membranewas washed thoroughly with de-ionized water for 24 hrs.

[0247] The mass gain of the resulting membrane after drying was 45.7 wt% and water flux was 7319±180 kg/m²h at 100 kPa.

[0248] The protein (lysozyme) absorption/desorption characteristics ofthe membrane were examined using the general procedure for a singlemembrane disk outlined earlier. A relatively steep break through curvewas observed. The membrane had a lysozyme binding capacity of 63.4mg/ml. The desorption experiment indicated that the recovery of proteinwas 79.3%.

Example 20

[0249] This example illustrates the effect of a neutral co-monomer onthe protein binding capacity of composite materials of this invention.TABLE 6 Chemical composition of stock solutions (amount of monomers in10 mL of a solution) Solvents (Dioxane/ DMF/ H₂O) Stock AAM AMPSPEG2000XL BIS Irgacure ® volumetric ID (g) (g) (g) (g) 2959, (g) ratioS1 0.60 0.40 1.00 0.25 0.01 8:1:1 S2 0.40 0.60 1.00 0.25 0.01 8:1:1 S30.20 0.80 1.00 0.25 0.01 8:1:1 S4 0   1.00 1.00 0.25 0.01 8:1:1

[0250] Monomer solutions were prepared by dilution of stock solutionsS1-S4 in Table 6 with acetone with the mass ratio of 1:1.

[0251] Composite membranes M1-M4 were prepared by using thecorresponding diluted solutions of stocks S1-S4 and following thegeneral preparation procedure described earlier. The porous support usedwas PP1545-4 and the irradiation time was 90 minutes. Upon completion ofpolymerization, the composite membranes were washed with de-ionizedwater for 24 hrs.

[0252] The properties and protein binding capacities of compositemembranes were examined and the results shown in Table 7. It is evidentthat the charge density of polyelectrolyte gels influences significantlyprotein adsorption onto membranes. TABLE 7 Properties and Lysozymeadsorption capacities of composite membranes Flux at Binding 100 kPaCapacity No. (kg/m² · h) (mg/ml) M1 8146 ± 96 56.9 M2 4273 ± 46 76.3 M3 7940 ± 303 41.5 M4 8651 ± 72 16.4

Example 21

[0253] This example illustrates the effect of the chain length of thepolyfunctional macromonomers used as cross-linkers (PEG diacrylates) onprotein binding capacity of composite materials of this invention.

[0254] A series of stock solutions containing 0.6 g of2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 0.4 g of acrylamide(AAM), 0.10 g of N,N′-methylenebisacrylamide (BIS), 0.01 g of Irgacure®2959, and 1.0 g of PEG diacrylate with different molecular weights(200,1000, 2000, 4000), obtained in Example 14, dissolved in 10 ml of adioxane-DMF-water mixture with a volume ratio 80:10:10, respectively,was prepared. The stock solutions were subsequently diluted with acetoneat the mass ratio of 1:1. A series of composite membranes were preparedfrom these solutions using poly(propylene) support PP1545-4 and byfollowing the general preparation procedure described above. Theirradiation time used was set to 90 minutes. Upon completion ofpolymerization, the composite membranes were washed with de-ionizedwater for 24 hrs.

[0255] The properties and protein binding capacities of compositemembranes were examined according to the general procedure for a singlemembrane disk. The results shown in Table 8 clearly indicate that thegel structure of composite membranes has substantial effect on proteinadsorption. Possibly, it is related to the gel structure near themacropore surface, where an extremely loose structure may be formed thatcan allow protein to penetrate into the gel layer at a certain depth.Another possibility is that by using a longer chain PEG diacrylate thesurface area is increased owing to some fuzziness at the surface andthus making more adsorption site available to proteins. TABLE 8Properties and Lysozyme adsorption capacities of composite membranesBinding PEG Flux at 100 kPa Capacity diacrylate (kg/m²h) (mg/ml)  2008390 ± 218 24.4 1000 7275 ± 139 58.1 2000 4273 ± 46  76.3 4000 6039 ±111 75.4

Example 22

[0256] This example illustrates the use of fibrous non-woven support toproduce a composite material of this invention containing positivelycharged macroporous gel.

[0257] A 10 wt-% solution was prepared by dissolvingdiallyldimethylammonium chloride (DADMAC) and acrylamide (AAM), whichwere taken in the ratio 80:20, in a solvent mixture containing 65 wt-%of dioxane, 18 wt-% of water, and 17 wt-% of DMF.N,N′-methylenebisacrylamide (BIS) was added to the monomer solution toobtain 40% (mol/mol) cross-linking degree. The photoinitiator Irgacure®2959 was added in the amount of 1% with respect to the total mass of themonomers.

[0258] A sample of the fibrous non-woven polypropylene substrate TR2611Awas placed on a polyethylene sheet and filled with the monomer solution.The substrate was subsequently covered with another polyethylene sheetand the resulting sandwich was run between two rubber rollers to pressthe monomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs. A duplicate sample was used to estimatethe mass gain of the composite material. The substrate gained 45% of theoriginal weight in this treatment.

[0259] The composite material produced by this method had a water fluxin the range of 2320 kg/m² hr at 70 kPa.

[0260] The protein (BSA) adsorption characteristic of a mono-layer ofthe composite material was examined using the general procedures one fora single membrane disk and one for a multi-membrane stack, as decribedabove. The membrane stack contained 7 membrane layers of total thickness1.75 mm. In both experiments the protein concentration was 0.4 g/L in a50 mM TRIS buffer solution, and the flow rate of the protein solutionused was 3.1±0.1 ml/min, delivered by peristaltic pump. The breakthroughcapacity for BSA was 64 mg/ml in the single membrane experiment and 55±2mg/ml in the multi-membrane stack experiment.

Example 23

[0261] This example illustrates the use of a mixture of two monomers inmaking a positively charged composite material of this invention.

[0262] A 10 wt-% solution was prepared by dissolvingdiallyldimethylammonium chloride (DADMAC) and(3-acrylamidopropyl)trimethylamonium chloride (APTAC), in the ratio50:50, in a solvent mixture containing 65 wt-% dioxane, 18 wt-% waterand 17 wt-% DMF. N,N′-methylenebisacrylamide (BIS) was added to themonomer solution to obtain 40% (mol/mol) cross-linking degree. Thephotoinitiator Irgacure® 2959 was added in the amount of 1% with respectto the total mass of the monomers.

[0263] A sample of the non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs.

[0264] The composite material produced by this method had a water fluxin the range of 2550 kg/m² hr at 70 kPa. The mass gain determined with aduplicate sample was found to be 45%.

[0265] The protein (BSA) adsorption characteristic of the mono-layercomposite material was examined using the general procedure for a singlemembrane disk described above. A solution of BSA concentration of 0.4g/L in a 50 mM TRIS buffer solution was delivered to the membrane at aflow rate of 2-4 ml/min. The breakthrough capacity of the compositematerial was 40 mg/ml.

Example 24

[0266] This example illustrates the effect of addition of a neutralmonomer to the mixture of charged monomers used in example 23.

[0267] A 15 wt-% solution was prepared by dissolvingdiallyldimethylammonium chloride (DADMAC),(3-acrylamido-propyl)trimethylammonium chloride (APTAC), and acrylamide(AAM), which were taken in the ratio 40:40:20, in a solvent mixturecontaining of 65 wt-% dioxane, 17 wt-% of water, and 18 wt-% of DMF.N,N′-methylenebisacrylamide (BIS) was added to the monomer solution toobtain 20% (mol/mol) cross-linking degree. The photoinitiator Irgacure®2959 was added in the amount of 1% with respect to the total mass of themonomers.

[0268] A sample of the non-woven polypropylene substrate TR2611A wasplaced on a polyethylene sheet and filled with the monomer solution. Thesubstrate was subsequently covered with another polyethylene sheet andthe resulting sandwich was run between two rubber rollers to press themonomer solution into the pores and remove excess of solution. Thefilled substrate was irradiated at 350 nm for 20 min for thepolymerization process to occur. The composite material was removed frombetween the polyethylene sheets, washed with water, TRIS-buffer solutionand stored in water for 24 hrs.

[0269] The composite material produced by this method had a water fluxof 550 kg/m² hr at 70 kPa and a mass gain (determined using a duplicatesamples) of 65 wt-%.

[0270] The protein (BSA) adsorption characteristic of the mono-layercomposite material was examined using the general procedure for a singlemembrane disk described above. A solution of BSA concentration of 0.4g/L in a 50 mM TRIS buffer solution was delivered to the membrane at aflow rate of 3.5-4 ml/min. The breakthrough capacity of the compositematerial was 130 mg/ml.

Example 25

[0271] This example illustrates the formation of unsupported positivelycharged macroporous gel by cross-linking of a preformed polymer.

[0272] A 10% solution of poly(allylamine hydrochloride) PAH was preparedby dissolving the polymer in a solvent mixture containing 60% water and40% iso-propanol (2-propanol). The polymer was partially deprotonated(40%) by adding 6.67 N NaOH. Ethylene glycol diglycidyl ether (EDGE) wasadded to this solution to obtain 40% (mol/mol) degree of cross-linking.The solution was kept at room temperature for 3 hours for gel formationby the cross-linking reaction between the amine groups of PAH and epoxygroups of EDGE. After 3 hours, the gel was placed in a water bath forall un-reacted chemicals to leach out.

[0273] A sample of the wet gel was examined using ESEM. The micrographshown in FIG. 14 indicates that a macroporous gel was formed with thepore diameter of about 70-80 μm. The wet gel was mechanically very weak.

Example 26

[0274] This example illustrates the making macroporous gel incorporatedin a non-woven fabric support.

[0275] A 10 wt-% solution of poly(allylamine hydrochloride) (PAH) wasprepared as in Example 25. The polymer was partially deprotonated andEDGE added as described in Example 25 and the solution was applied to asample of the non-woven polypropylene membrane support TR2611A placedbetween two polyethylene sheets. The resulting sandwich was run betweentwo rubber rollers to press the polymer solution into the pores of thesubstrate, spread it evenly, and remove the excess solution. Thesolution-filled support sample was kept at room temperature for 3 hoursfor cross-linking process to take place resulting in the formation ofgel. After that time, the composite material was removed from thesandwich and placed in a water bath for 12 hours to leach out unreactedchemicals.

[0276] A wet sample of the resulting composite membrane was examinedusing ESEM. The micrograph shown in FIG. 15 indicates the compositemembrane having macroporous gel in the fibrous non-woven support member.The average pore size of the gel was about 25-30 μm. The membranethickness was 800 μm and the water flux measured at 100 kPa was 592kg/m²h. The composite material showed rather low BSA binding capacity ofabout 10 mg/ml.

Example 27

[0277] This example provides a comparison of the protein adsorption by acomposite membrane of this invention with the commercial Mustang® Coin Qproduced by Pall Corporation.

[0278] A composite material prepared in example 22 was tested in amulti-membrane stack of 7 membrane layers of a total thickness of 1.75mm, according to the testing protocol described in example 22. AMustang® Coin Q was also tested under similar conditions. The membranestack was prepared by placing seven (7) layers of the, membrane samplein the wet state on top of each other. The assembled membrane stack waslightly compressed pressed to remove excess of water. The membrane stackwas then heated in an oven at 60-70° C. for at least 30 min. Thethickness of the resulting membrane stack in the dry state was 1.8-1.9mm. This process produced a stack in which the multiple membrane layersadhered to each other. The results shown in FIG. 16 indicate that bothsystems give similar performances.

Example 28

[0279] This example provides the hydrodynamic (Darcy) permeability ofreference composite materials containing porous support member andhomogeneous gels filling the pores of the support. The homogeneous gelswere obtained by using thermodynamically good solvents and theirhomogeneity was assessed based on transparency of simultaneously formedunsupported gels of the same composition. Clear and transparent gelswere assumed to be homogeneous, contrary to macroporous gels that werealways found opaque.

[0280] (A) Glycidyl Methacrylate Based Homogeneous Gel-Filled Composites

[0281] The composite materials containing homogeneous gels of glycidylmethacrylate-co-ethylene glycol dimethacrylate, GMA-co-EDMA, wereprepared using 1,4-dioxane as a solvent and 4.7 wt-% of EDMA(cross-linker) in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 and the generalprocedure for preparing the composite materials of this invention wereused. DMPA was used as photoinitiator and the irradiation was carriedout at 350 nm for 120 minutes. The hydrodynamic permeability of themembranes was measured and an empirical equation was derived for therelationship between the hydrodynamic permeability, k, and the mass gainof the composite membranes containing poly(GMA-co-EDMA) homogeneous gelsin the PP1545-4 support. The equation is as follows:

k=3.62×10³ ×G ^(−9.09)

[0282] The differences between the measured values of permeability andthat calculated from the above equation were found to be less than ±3%.This empirical relationship was subsequently used to calculatepermeability of reference composite materials at different mass gains.

[0283] (B) Poly(diallyldimethylammonium Chloride) Based HomogeneousGel-Filled Composites

[0284] The composite materials containing homogeneous gels ofdiallyldimethylammonium chloride-co-methylenebisacrylamide,DADMAC-co-BIS, were prepared using water as a solvent and 1.0 wt-% ofBIS cross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the compositematerials of this invention was used to make homogeneous gel-filledmembranes. Irgacure® 2959 was used as photoinitiator and the irradiationwas carried out at 350 nm for 30-40 minutes. The hydrodynamicpermeability of the series of membranes was measured and an empiricalequation was derived for the relationship between the Darcypermeability, k, and the mass gain, G:

k=2.09×10⁻¹² ×G ^(−4.01)

[0285] (C) Acrylamide Based Homogeneous Gel-Filled Composites

[0286] The hydrodynamic permeability of homogeneouspoly(acrylamide)-co-methylenebisacrylamide, AAM-co-BIS, was estimatedfrom the empirical relationship between the gel permeability and the gelpolymer volume fraction provided by Kapur et al. in Ind. Eng. Chem.Res., vol. 35 (1996) pp. 3179-3185. According to this equation, thehydrodynamic permeability of a poly(acrylamide) gel,k_(gel)=4.35×10⁻²²×φ^(−3.34), where φ is the polymer volume fraction inthe gel. In the same article, Kapur et al. provide a relationshipbetween the hydrodynamic permeability of a gel and a porous membranefilled with the same gel. According to this relationship, thepermeability of the membrane, k_(membrane)=(ε/τ)×k_(gel), where ε is theporosity of the support and τ is the tortuosity of the support pores.The pore tortuosity can be estimated as a ratio of the Kozeny constant,K, for a given porosity, i.e., K=5, and the Kozeny constant for astraight cylindrical capillary equal to 2. Thus, for the poly(propylene)support PP1545-4 with porosity of 0.85, the ratio (ε/τ)=0.85/2.5=0.34.

[0287] The polymer volume fraction, φ, can be converted to mass gainusing the partial specific volume, ν₂, for poly(acrylamide) and thedensity, ρ, of poly(propylene). The values of these parameters can befound in Polymer Handbook, edited by Brandrup et al., Chapter VII, Wileyand Sons, New York, 1999. Thus, the mass gain of a composite materialcontaining poly(propylene) support of porosity ε filled with a gel whosepolymer occupies the fraction φ of the pores is given by${{Mass}\quad {Gain}\quad (\%)} = {\frac{\phi/v_{2}}{\left( {1 - ɛ} \right)\quad \rho} \times 100\%}$

[0288] The above equation was combined with that of Kapur et al. to givean empirical relationship allowing one to calculate hydrodynamicpermeability of reference composite materials, k, at different massgains, G. The combined equation is as follows:

k=1.80×10⁻¹² ×G ^(−3.34)

[0289] The equation is valid for ρ=0.91 g/cm³; ε=0.85; ν₂=0.7 cm³/g;(ε/τ)=0.34.

[0290] (D) Poly(AMPS) Based Homogeneous Gel-Filled Composites

[0291] The composite materials containing homogeneous gels of2-acrylamido-2-propane-1-sulfonic acid-co-methylenebisacrylamide,AMPS-co-BIS, were prepared using water as a solvent and 10.0 wt-% of BIScross-linker in monomer mixture, and different total monomerconcentrations. Poly(propylene) support PP1545-4 was coated with TritonX-114 surfactant by immersing the support samples in 2t-% solution ofthe surfactant in methanol/water (60/40) mixture for 16 hours followedby drying in air. The general procedure for preparing the compositematerials of this invention was used to make homogeneous gel-filledmembranes. Irgacure® 2959 was used as photoinitiator and the irradiationwas carried out at 350 nm for 60 minutes. The hydrodynamic permeabilityof the series of membranes was measured and an empirical equation wasderived for the relationship between the Darcy permeability, k, and themass gain, G:

k=2.23×10⁻¹⁶ ×G ^(−1.38)

[0292] (E) Poly(APTAC) Based Homogeneous Gel-Filled Composites

[0293] The composite materials containing homogeneous gels of(3-acrylamidopropane)trimethylammoniumchloride-co-methylenebisacrylamide, APTAC-co-BIS, were prepared usingwater as a solvent and 10.0 wt-% of BIS cross-linker in monomer mixture,and different total monomer concentrations. Poly(propylene) supportPP1545-4 was coated with Triton X-114 surfactant by immersing thesupport samples in 2t-% solution of the surfactant in methanol/water(60/40) mixture for 16 hours followed by drying in air. The generalprocedure for preparing the composite materials of this invention wasused to make homogeneous gel-filled membranes. Irgacure® 2959 was usedas photoinitiator and the irradiation was carried out at 350 nm for 60minutes. The hydrodynamic permeability of the series of membranes wasmeasured and an empirical equation was derived for the relationshipbetween the Darcy permeability, k, and the mass gain, G:

k=9.5×10⁻¹⁶ ×G ^(−1.73)

[0294] (F) Poly(ethyleneimine) Based Homogeneous Gel-Filled Composites

[0295] The composite materials containing homogeneous gels of branchedpoly(ethyleneimine) cross-linked with ethylene glycol diglycidyl ether(EDGE) were prepared using methanol solutions of BPEI of differentconcentrations. The degree of cross-linking used was 10 mol-%.Poly(propylene) support PP1545-4 was used together with the generalprocedure of fabrication of composite materials by in situ cross-linkingof cross-linkable polymers described in Example 10. A series ofmembranes with different mass gains was prepared by changing theconcentration of PEI in the solution. The Darcy permeability of themembranes was measured and an empirical equation describing therelationship between the permeability, k, and the mass gain, G, wasderived. The equation is as follows:

k=4.38×10⁻¹⁴ ×G ^(−2.49)

Example 29

[0296] This example provides comparison between Darcy permeability ofcomposite materials of this invention containing supported macroporousgels and the permeability of the reference composite materialscontaining homogeneous gels filling the porous support member used inthis invention.

[0297] The comparison is shown in Table 9 below. TABLE 9 PermeabilityRatio Values for Composite Materials Composite Materials Darcycontaining Macroporous Gels Permeability of this Invention of ReferenceDarcy Composite Permeability Permeability Material Ratio Example Massk_(macroporous) k_(homogeneous) k_(macroporous)/ No. Gain % m² m²k_(homogeneous) 2 107 9.53 × 10⁻¹⁶ 2.93 × 10⁻¹⁹ 3.2 × 10³ 5  74 1.58 ×10⁻¹⁵ 2.49 × 10⁻¹⁸ 6.3 × 10² 6  92* 1.98 × 10⁻¹⁸* 1.85 × 10⁻¹⁸* 1.1 ×10⁰* 100 3.53 × 10⁻¹⁶ 1.65 × 10⁻¹⁸ 2.2 × 10²  100* 5.19 × 10⁻¹⁸* 1.65 ×10⁻¹⁸* 3.2 × 10⁰* 7 103  4.4 × 10⁻¹⁵ 1.58 × 10⁻¹⁸ 2.8 × 10³ 8  42 9.87 ×10⁻¹⁶ 8.81 × 10⁻¹⁹ 1.1 × 10³ 9  80 1.09 × 10⁻¹⁶ 6.78 × 10⁻²⁰ 1.6 × 10³10  95 1.89 × 10⁻¹⁵ 5.40 × 10⁻¹⁹ 3.5 × 10³ 11 144 2.77 × 10⁻¹⁵ 8.39 ×10⁻¹⁹ 3.3 × 10¹ 158 9.15 × 10⁻¹⁶ 3.77 × 10⁻¹⁷ 2.4 × 10¹ 237 1.66 × 10⁻¹⁷9.20 × 10⁻¹⁹ 1.8 × 10¹ 265 2.48 × 10⁻¹⁵ 3.34 × 10⁻¹⁹ 7.5 × 10³ 277 6.96× 10⁻¹⁶ 2.24 × 10⁻¹⁹ 3.1 × 10³ 12 103 1.59 × 10⁻¹⁸* 3.38 × 10⁻¹⁹* 4.7 ×10⁰* 108 2.52 × 10⁻¹⁸* 2.93 × 10⁻¹⁹* 8.6 × 10⁰* 110 2.11 × 10⁻¹⁸* 2.70 ×10⁻¹⁹* 7.8 × 10⁰* 112 9.32 × 10⁻¹⁸ 2.61 × 10⁻¹⁹ 3.6 × 10¹ 130 5.34 ×10⁻¹⁶ 1.56 × 10⁻¹⁹ 3.4 × 10³ 273 8.57 × 10⁻¹⁷ 1.31 × 10⁻²⁰ 6.5 × 10³ 3073.88 × 10⁻¹⁷ 8.87 × 10⁻²¹ 4.4 × 10³ 15 113 2.26 × 10⁻¹⁶ 1.39 × 10⁻¹⁸ 1.6× 10² 16  51 3.73 × 10⁻¹⁵ 4.16 × 10⁻¹⁸ 9.0 × 10² 17  34 7.52 × 10⁻¹⁵7.18 × 10⁻¹⁸ 1.0 × 10³ 18 108 6.47 × 10⁻¹⁶ 1.47 × 10⁻¹⁸ 4.4 × 10² 19  464.52 × 10⁻¹⁵ 4.85 × 10⁻¹⁸ 9.3 × 10²

Examples 30-37

[0298] These examples illustrate a method of preparing a responsivecomposite material of the present invention using photoinitiated freeradical polymerization of acrylic acid (AA) (ionic monomer), acrylamide(AA), and trimethylolpropane triacrylate (TRIM) as a cross-linker. Themolar ratio of acrylic acid to acrylamide was 1:1 and 1,4-dioxane wasused as a solvent in all experiments. Monomer solution compositions andpolymerization conditions are given in Table 10. After polymerization,the responsive composite material was washed with de-ionized water forabout 16 hrs. TABLE 10 Monomer solution compositions and polymerizationconditions Total Concentration of Monomer Degree of Irradiation ExampleSupport Mixture Cross-linking Time Mass Gain no. Sample ID Member (wt-%)(mol-%) (min) (%) 30 AM675 TR2611A 19.9 5.0 20 115.6 31 AM678 TR2611A13.3 5.0 90 81.7 32 AM680 TR2611A 12.8 5.2 20 82.3 33 AM681 TR2611A 12.610.8 15 88.8 34 AM682 TR2611A 23.8 10.9 15 167.3 35 AM684 TR2611A 31.010.8 10 217.3 36 AM683 TR2611A 38.8 10.8 15 294.6 37 AM694 PP 1545-424.0 10.2 10 218.0

[0299] The amount of gel formed in the support member depends on thepore volume available to fill, the total concentration of the monomermixture, and the degree of conversion in the polymerization. In FIG. 17,the mass gain obtained with support TR2611A is plotted as a function oftotal monomer concentration. The data can be approximated to fall withina straight line (R²=0.97), indicating a similar degree of conversion foreach sample. The experimental values are very close to their theoreticalcounterparts estimated from the pore volume in the support and themonomer concentration. This suggests that the degree of conversion isclose to 100% and that an irradiation time of 10 minutes is sufficientunder the light conditions applied. As expected, the mass gain obtainedwith the PP 1545-4 support was higher than that obtained with theTR2611A support due to the larger porosity of the former (85 vol-%versus 79.5 vol-%).

Example 38

[0300] This example illustrates the responsiveness of the responsivecomposite materials according to Example 30 to ionic interactions. Forthis purpose, the composite materials were tested with solutions ofdifferent pH and/or salt concentrations by measuring the flux at 100kPa. A typical change in flux taking place with the change of pH fromabout 3 (1 mM HCl) to about 12 (1 mM NaOH), obtained with membrane AM675is shown in FIG. 18. It can be seen from the Figure that the fluxmeasured with 1 mM HCl is nearly 100 times larger than the flux measuredwith 1 mM NaOH. The reason for this behavior of the membranes lies inchanges in the degree of ionization of the acid component of themacroporous gel. At high pH (1 mM NaOH) the carboxyl groups of the acidcomponent become ionized and the electrostatic repulsive force causesthe polymer chains to uncoil and stretch until balanced by counteractingforces of the polymer network elasticity and confinement imposed by thesupport member of the membrane. The swelling polymer chains reduce thepore volume and the pore radius in the gel. At low pH (1 mM HCl), thecarboxyl groups are converted into neutral carboxylic acid groups, theelectrostatic forces disappear, and the gel shrinks (collapses)enlarging the pores in the gel. The presence of the support memberprevents the gel from collapsing as a whole, i.e., from the process thatwould occur in the unsupported bulk gel, and closing the pores. Thus,the presence of the support reverses the direction in which hydraulicproperties of the gel change. When pure water flux is measured, thevalues obtained depend on the distance from equilibrium ionization atthe water pH (˜5.5). The initial water flux can be assumed to bemeasured at equilibrium. Immediately after the acid or base, the gel isfar from equilibrium with water and the pure water flux reflects thisstate by being close to the flux in the ionized form (after NaOH) orneutral form (after HCl).

[0301] The ratio of the flux measured with 1 mM HCl to that measuredwith 1 mM NaOH has been taken as a measure of membrane response (MR).The results obtained with membranes described in Example 30-37 are shownin Table 11. TABLE 11 Results for Examples 30-37 Membrane ID AM675 AM678AM680 AM681 AM682 AM684 AM683 AM694 Total 19.9 13.3 12.8 12.6 23.8 31.038.8 24.0 Monomer Conc. Wt-% Degree 5.0 5.0 5.2 10.8 10.9 10.8 10.8 10.2of Cross- linking Mol-% Membrane 89.6 372.2 352.0 29.4 10.3 5.6 4.8 19.5Response (MR)

[0302] The results in Table 11 show that the response of the compositemembranes of this invention to ionic interaction can also be controlledby the total concentration of monomer mixture and the degree ofcross-linking. As the monomer concentration increases, the membranesensitivity to the environmental changes decreases. Similar effect isfound when the degree of cross-linking is increased.

Example 39

[0303] This example illustrates the ability of membranes based onresponsive composite materials of this invention to fractionateproteins. The separation of therapeutic proteins Human Serum Albumin(HSA) and Human Immunoglobulin G (HIgG) was chosen as a case study.Human plasma is the starting material for the production of a number oftherapeutic proteins, which are referred to as plasma proteins. The mostabundant amongst these are HSA and HIgG, both of which are manufacturedin bulk quantities. These proteins are generally fractionated byprecipitation based processes which give high product throughput butpoor resolution in terms of separation. Membrane based processes such asultrafiltrations have the potential for giving both high throughput andhigh resolution.

[0304] Two composite membranes of this invention containing responsivemacroporous gel, duplicates of membrane AM695 (see Tables 10 and 11),were tested for their suitability in separation of these plasmaproteins. In the experiments discussed here, the change in membrane poresize with change in salt concentration was utilized to effectprotein-protein separation in the manner desirable, i.e. sequentialrelease from the membrane module. Other environmental conditions such aspH could well be used to achieve a similar objective.

[0305] A binary carrier phase system was used in the ultrafiltrationexperiments. The starting carrier phase in all the experiments was onewith a low salt concentration (typically 5-10 mM NaCl). In all theexperiments the carried phase was switched to one with a high saltconcentration (typically 1 M NaCl). The change in salt concentrationwithin the membrane module could be tracked by observing theconductivity of the permeate stream. The change in transmembranepressure gave an idea about the change in membrane hydraulicpermeability with change in salt concentration. FIG. 19 shows thechanges of transmembrane pressure and conductivity as a function of thepermeate salt concentration (FIGS. 19. A and B) and the changes oftransmembrane pressure as a function of permeate conductivity (FIG.19C). In this experiment the salt concentration was being increasedcontinuously in a linear fashion. The transmembrane pressure observed isrelated to the permeate salt concentration and reflects changes inpore-diameter.

[0306] Experiments were carried out using human serum albumin and humanimmunoglobulin mixtures. The ultrafiltration was started at a low saltconcentration (i.e. 10 mM). At this condition, human serum albumin wastransmitted while human immunoglobulin G was almost totally retained.The salt concentration was then increased and this increased the porediameter (as evident from drop in pressure in constant permeate fluxultrafiltration). This in turn led to the transmission of humanimmunoglobulin G through the membrane. Hence by altering theenvironmental condition it was possible to sequentially transmitproteins having different sizes through the same membrane. If theinitial mixture had also contained a protein significantly larger thanhuman immunoglobulin, it would have been possible to fractionate thethree proteins (i.e. human serum albumin, human immunoglobulin and thesignificantly bigger protein) by appropriately controlling the change insalt concentration. Two of the three fractions obtained here would be inthe permeate while the third fraction would be in the retentate.

[0307] The results obtained with duplicates of membrane AM694 are shownin FIGS. 20, 21, and 22. FIG. 20 shows the results obtained with HIgGultrafiltration. As evident from the figure, very little, if any HIgGwas transmitted at the low salt concentration. However, when the saltconcentration was increased, the HIgG was released from the membranemodule. The drop in TMP with increase in salt concentration was due tothe increase in pore diameter.

[0308] The results presented in FIG. 21 were obtained with HSAultrafiltration. As evident from the figure HSA was freely transmittedthrough the membrane even at low salt concentration. When the saltconcentration was increased, the transmission of HSA was found toincrease a bit.

[0309]FIG. 22 shows the results obtained with HSA/HIgG ultrafiltration.At low salt concentration, HSA alone was transmitted. Ultrafiltrationwas continued until HSA was nearly completely removed from the membranemodule. The HIgG was then released by increasing the salt concentration.

Example 40

[0310] This example illustrates a method for making a positively chargedcomposite material of the invention having a high protein bindingcapacity.

[0311] A 15 wt-% solution was prepared by dissolving(3-acrylamidopropyl)-trimethylammonium chloride (APTAC),N-(hydroxymethyl)acrylamide and N,N′-methylenebisacrylamide ascross-linker in a molar ratio of 1:0.32:0.1, respectively, in a solventmixture containing 10 wt-% water, 60 wt-% di(propylene glycol)methylether and 30 wt-% dimethylformamide (DMF). The photo-initiator Irgacure®2959 was added in the amount of 1% with respect to the mass of themonomers.

[0312] A sample of the fibrous non-woven polypropylene substrate TR2611Awas placed on a polyethylene sheet and filled with the monomer solution.The substrate was subsequently covered with another polyethylene sheetand the resulting sandwich was run between two rubber rollers to pressthe monomer solution into the pores and to remove any excess solution.The substrate was irradiated for 5 minutes at 350 nm. The compositematerial was then removed from between the polyethylene sheets, washedwith water and TRIS-buffer solution and stored in water for 24 hrs.

[0313] Several samples were prepared according to the above process, andthe samples were then dried and weighed. The average mass gain of thecomposite material was 55.7% of the original weight of the startingsupport member.

[0314] The protein (BSA) adsorption characteristic of a multi-membranestack of the above composite material was examined using the generalprocedure for a mono-layer of the composite material, as describedearlier. The membrane stack tested contained 4 membrane layers, giving atotal thickness 1.05 mm. The protein solution used was a 25 mM TRISbuffer solution with a protein concentration of 0.4 g/L, and the flowrate of the protein solution was 5.0 ml/min at 150 kPa. The breakthroughcapacity for BSA was 281 mg/ml. In a subsequent desorption step,approximately 85% of the BSA was recovered.

[0315] All references mentioned herein are incorporated herein byreference to the same extent as if each reference were stated to bespecifically incorporated herein by reference.

[0316] To those skilled in the art, it is to be understood that manychanges, modifications and variations could be made without departingfrom the spirit and scope of the present invention as claimedhereinafter.

1. A composite material that comprises a support member that has aplurality of pores extending through the support member and, located inthe pores of the support member, and filling the pores of the supportmember, a macroporous cross-linked gel.
 2. A composite materialaccording to claim 1 that has a permeability ratio of at least
 10. 3. Acomposite material according to claim 1 that has a permeability ratio ofat least
 100. 4. A composite material according to claim 1 that has apermeability ratio of at least
 1000. 5. A composite material accordingto claim 1, wherein the macroporous gel is non-self supporting.
 6. Acomposite material according to claim 1, wherein the macroporouscross-linked gel has macropores of average size between approximately 10and approximately 3000 nm and a volume porosity between 30 and 80%.
 7. Acomposite material according to claim 1, wherein the macroporouscross-linked gel has macropores of average size between 25 and 1500 nm.8. A composite material according to claim 1, wherein the macroporouscross-linked gel has macropores of average size between 50 and 1000 nm.9. A composite material according to claim 1, wherein the macroporouscross-linked gel has macropores of average size of about 700 nm.
 10. Acomposite material according to claim 1 in the form of a membrane foruse as a filter in size exclusion separation.
 11. A composite materialaccording to claim 1 in the form of a membrane and wherein themacroporous cross-linked gel bears charged moieties.
 12. A compositematerial according to claim 1 in the form of a membrane and in which themacroporous cross-linked gel bears a ligand for attachment of biologicalmolecules, biological ions, or both.
 13. A composite material accordingto claim 1, wherein the support member has a void volume that is notcompletely occupied by the macroporous gel, and the density of themacroporous gel is greater at or adjacent to a first major surface ofthe support member than the density at or adjacent to a second majorsurface of the support member.
 14. A composite material according toclaim 1, wherein the support member has a void volume that issubstantially completely occupied by the macroporous gel.
 15. Acomposite material according to claim 1, wherein the support member ismade of polymeric material in the form of a membrane that has athickness of from between about 10 μm to about 500 μm, contains pores ofaverage size between 0.1 to 25 μm, and has a volume porosity between 40and 90%.
 16. A composite material according to claim 1, wherein thesupport member is made of an extended polyolefin formed by phaseseparation.
 17. The composite material according to claim 1, wherein thesupport member comprises a polymeric material selected from the groupconsisting of polysulfones, polyethersulfones, polyphenyleneoxides,polycarbonates, polyesters, cellulose and cellulose derivatives.
 18. Acomposite material according to claim 1, wherein the support member ismade of polymeric material in the form of a fibrous woven or non-wovenfabric that has a thickness of from between about 10 μm to about 2000μm, contains pores of average size between 0.1 to 25 μm, and has avolume porosity between 40 and 90%.
 19. A composite material accordingto claim 1, wherein the support member comprises a stack of 2 to 10separate support members.
 20. A composite material according to claim 1,wherein the macroporous cross-linked gel is a neutral or chargedhydrogel, a polyelectrolyte gel, a hydrophobic gel, a neutral gel, or afunctional gel.
 21. A composite material according to claim 20, whereinthe neutral or charged hydrogel is selected from the group consisting ofcross-linked poly(vinyl alcohol), poly(acrylamide),poly(isopropylacrylamide), poly(vinylpyrrolidone), poly(hydroxymethylacrylate), poly(ethylene oxide), copolymers of acrylic acid ormethacrylic acid with acrylamide, isopropylacrylamide, orvinylpyrrolidone, copolymers of acrylamide-2-methyl-1-propanesulfonicacid with acrylamide, isopropylacrylamide, or vinylpyrrolidone,copolymers of (3-acrylamidopropyl)trimethylammonium chloride withacrylamide, isopropylacrylamide, or N-vinylpyrrolidone, and copolymersof diallyldimethylammonium chloride with acrylamide,isopropylacrylamide, or vinylpyrrolidone.
 22. A composite materialaccording to claim 20, wherein the polyelectrolyte gel is selected fromthe group consisting of cross-linkedpoly(acrylamido-2-methyl-1-propanesulfonic acid) and its salts,poly(acrylic acid) and its salts, poly(methacrylic acid) and its salts,poly(styrenesulfonic acid) and its salts, poly(vinylsulfonic acid) andits salts, poly(alginic acid) and its salts,poly[(3-acrylamidopropyl)trimethylammonium] salts,poly(diallyldimethylammonium) salts, poly(4-vinyl-N-methylpyridinium)salts, poly(vinylbenzyl-N-trimethylammonium) salts, andpoly(ethyleneimine) and its salts.
 23. A composite material according toclaim 20, wherein the hydrophobic gel is selected from the groupconsisting of cross-linked polymers or copolymers of ethyl acrylate,n-butyl acrylate, propyl acrylate, octyl acrylate, dodecyl acrylate,octadecylacrylamide, stearyl acrylate, and styrene.
 24. A compositematerial according to claim 20, wherein the neutral gel is selected fromthe group consisting of cross-linked polymers or copolymers ofacrylamide, N,N-dimethylacrylamide, N-methacryloylacrylamide,N-methyl-N-vinylacetamide, and N-vinylpyrrolidone.
 25. A compositematerial according to claim 20, wherein the functional gel hasfunctional groups that take the form of antibodies, amino acid ligands,antigen and antibody ligands, dye ligands, and metal affinity ligands.26. A composite material according to claim 1, wherein the macroporouscross-linked gel comprises a macromonomer.
 27. A composite materialaccording to claim 26, wherein the macromonomer is selected from thegroup consisting of poly(ethylene glycol) acrylate and poly(ethyleneglycol)methacrylate.
 28. A composite material according to claim 1,wherein the macroporous cross-linked gel is cross-linked by apolyfunctional macromonomer.
 29. A composite material according to claim28, wherein the polyfunctional macromonomer is selected from the groupconsisting of poly(ethylene glycol)diacrylate and poly(ethyleneglycol)dimethacrylate.
 30. A composite material according to claim 1,wherein the macroporous cross-linked gel is a positively chargedhydrogel comprising α-co-polymer of(3-acrylamidopropyl)-trimethylammonium chloride (APTAC) andN-(hydroxymethyl)acrylamide cross-linked by N,N′-methylenebisacrylamide.31. A composite material according to claim 1, wherein the macroporouscross-linked gel is a responsive macroporous cross-linked gel.
 32. Acomposite material according to claim 31, wherein the responsivemacroporous cross-linked gel can be in: a) a stable collapsed state,wherein the average size of the macropores in the gel is from 10 to 2000nm, b) a stable swollen state, wherein the average size of themacropores in the gel is from 2 to 200 nm, or c) one of multiple stableintermediate states, wherein the average size of the macropores in thegel is between 2 and 2000 nm.
 33. A composite material according toclaim 31, wherein the responsive macroporous cross-linked gel isresponsive to variations in at least one of pH, ionic strength,temperature, light intensity, or electrochemical current.
 34. Acomposite material according to claim 31, wherein the responsivemacroporous cross-linked gel comprises a responsive monomer, a neutralmonomer and a cross-linking agent.
 35. A process for size-exclusionfiltration which comprises passing a liquid to be filtered through acomposite material according to claim
 1. 36. A process according toclaim 35, wherein the liquid is a suspension of cells or a suspension ofaggregates.
 37. A process for Donnan exclusion separation whichcomprises passing a liquid containing a charged material through acomposite material according to claim 1, which composite material bearscharges on the macroporous gel.
 38. A process for adsorbing a biologicalmolecule or a biological ion from a liquid, which comprises passing aliquid containing the biological molecule or biological ion through acomposite material according to claim 1, which composite material bearsbinding sites that display specific interactions for the biomolecule onthe macroporous gel.
 39. A process according to claim 38, wherein thebiological molecule or the biological ion is selected from the groupconsisting of albumins, lysozyme, viruses, cells, γ-globulins of humanand animal origins, immunoglobulins of both human and animal origins,proteins of recombinant or natural origin including, polypeptides ofsynthetic or natural origin, interleukin-2 and its receptor, enzymes,monoclonal antibodies, trypsin and its inhibitor, cytochrome C,myoglobulin, recombinant human interleukin, recombinant fusion protein,nucleic acid derived products, DNA of either synthetic or naturalorigin, and RNA of either synthetic or natural origin.
 40. A processaccording to claim 38, wherein the specific interactions areelectrostatic interactions.
 41. A process according to claim 38, whereinthe specific interactions are affinity interactions.
 42. A processaccording to claim 38, wherein the specific interactions are hydrophobicinteractions.
 43. A process for solid phase chemical synthesis whichcomprises passing a liquid, having a first reactant through a compositematerial according to claim 1, wherein a second reactant is in amacropore of the composite material.
 44. A process for preparing acomposite material that comprises a support member that has a pluralityof pores extending through the support member and, located in the poresof the support member and filling the pores of the support member, amacroporous cross-linked gel, the process comprising: a) introducinginto the pores of the support member a solution or suspension of i) oneor more monomers and one or more cross-linking agents that can combineto form a macroporous gel, or ii) one or more cross-linkable polymersand one or more cross-linking agents that can combine to form amacroporous gel, b) reacting the monomers and the cross-linking agent orthe polymer and the cross-linking agent to form a macroporouscross-linked gel that fills the pores of the support member.
 45. Aprocess according to claim 44, wherein the molar ratio of cross-linkingagent to monomers is in the range of from about 5:95 to about 70:30. 46.A process according to claim 44, wherein the molar ratio ofcross-linking agent to monomers is in the range of from about 10:90 toabout 50:50.
 47. A process according to claim 44, wherein the molarratios of cross-linking agent to monomers is in the range of from about15:85 to about 45:55.
 48. A process according to claim 44, wherein themonomer is selected from the group consisting of acrylamide,2-acryloxyethyltrimethylammonium chloride, N-acryloxysuccinimide,N-acryloyltris(hydroxymethyl)methylamine, 2-aminoethyl methacrylatehydrochloride, N-(3-aminopropyl)methacrylamide hydrochloride, butylacrylate and methacrylate, N,N-diethylacrylamide,N,N-dimethylacrylamide, 2-(N,N-dimethylamino)ethyl acrylate andmethacrylate, N-[3-(N,N-dimethylamino)propyl]methacrylamide,N,N-dimethylacrylamide, n-dodecyl acrylate, n-dodecyl methacrylate,dodecyl methacrylamide, ethyl methacrylate, 2-(2-ethoxyethoxy)ethylacrylate and methacrylate, 2,3-dihydroxypropyl acrylate andmethacrylate, glycidyl acrylate and methacrylate, n-heptyl acrylate andmethacrylate, 1-hexadecyl acrylate and methacrylate, 2-hydroxyethylacrylate and methacrylate, N-(2-hydroxypropyl)methacrylamide,hydroxypropyl acrylate and methacrylate, methacrylamide, methacrylicanhydride, methacryloxyethyltrimethylammonium chloride,2-(2-methoxy)ethyl acrylate and methacrylate, octadecyl acrylamide,octylacrylamide, octyl methacrylate, propyl acrylate and methacrylate,N-iso-propylacrylamide, stearyl acrylate, styrene, 4-vinylpyridine,vinylsulfonic acid, N-vinyl-2-pyrrodinone, particularly preferredmonomers include dimethyldiallylammonium chloride,acrylamido-2-methyl-1-propanesulfonic acid (AMPS),(3-acrylamidopropyl)trimethylammonium chloride (APTAC), acrylamide,methacrylic acid (MAA), acrylic acid (AA), 4-styrenesulfonic acid andits salts, acrylamide, glycidyl methacrylate, diallylamine, anddiallylammonium chloride. and sodium styrenesulfonate.
 49. A processaccording to claim 44, wherein the monomer is selected from the groupconsisting of dimethyldiallylammonium chloride,acrylamido-2-methyl-1-propanesulfonic acid (AMPS), (3-acrylamidopropyl)trimethylammonium chloride (APTAC), acrylamide, methacrylic acid (MAA),acrylic acid (AA), 4-styresulfonic acid and its salts, acrylamide,hydroxyalkylacrylamides, glycidyl methacrylate, diallylamine, anddiallylammonium chloride.
 50. A process according to claim 44, whereinone or more monomers are macromonomers.
 51. A process according to claim50, wherein the macromonomers have a molecular weight greater than about200 Da.
 52. A process according to claim 50, wherein the macromonomersare poly(ethylene glycol) acrylate or poly(ethylene glycol)methacrylate.53. A process according to claim 50, wherein the cross-linking agent isselected from the group consisting of bisacrylamidoacetic acid,2,2-bis[4-(2-acryloxyethoxy)phenyl]propane,2,2-bis(4-methacryloxyphenyl)propane, butanediol diacrylate anddimethacrylate, 1,4-butanediol divinyl ether, 1,4-cyclohexanedioldiacrylate and dimethacrylate, 1,10-dodecanediol diacrylate anddimethacrylate, 1,4-diacryloylpiperazine, diallylphthalate,2,2-dimethylpropanediol diacrylate and dimethacrylate, dipentaerythritolpentaacrylate, dipropylene glycol diacrylate and dimethacrylate,N,N-dodecamethylenebisacrylamide, glycerol trimethacrylate, glyceroltris(acryloxypropyl) ether, N,N′-hexamethylenebisacrylamide,N,N′-octamethylenebisacrylamide, 1,5-pentanediol diacrylate anddimethacrylate, 1,3-phenylenediacrylate, poly(ethylene glycol)(n)diacrylate and dimethacrylate, poly(propylene)(n) diacrylate anddimethacrylate, triethylene glycol diacrylate and dimethacrylate,triethylene glycol divinyl ether, and tripropylene glycol diacrylate anddimethacrylate, trimethylolpropane triacrylate, and divinylbenzene. 54.A process according to claim 44, wherein the cross-linking agent isselected from the group consisting of N,N,-methylenebisacrylamide,diethylene glycol diacrylate and dimethacrylate, ethylene glycoldiacrylate and dimethacrylate, tetra(ethylene glycol)diacrylate,1,6-hexanediol diacrylate, divinylbenzene, trimethylolpropanetriacrylate, and poly(ethylene glycol) diacrylate.
 55. A processaccording to claim 44, wherein the cross-linking agent is apolyfunctional macromonomer.
 56. A process according to claim 55,wherein the polyfunctional macromonomer has molecular weight greaterthan about 200 Da.
 57. A process according to claim 56, wherein thepolyfunctional macromonomer is poly(ethylene glycol) diacrylate orpoly(ethylene glycol)dimethacrylate.
 58. A process according to claim44, wherein the cross-linkable polymer is selected from the groupconsisting of poly(ethyleneimine), poly(4-vinylpyridine),poly(vinylbenzyl chloride), poly(diallylammonium chloride),poly(glycidyl methacrylate), copolymers of vinylpyridine anddimethyldiallylammonium chloride, and copolymers of vinylpyridine,dimethyladiallylammonium chloride, or(3-acrylamidopropyl)trimethylammonium chloride with glycidylmethacrylate.
 59. A process according to claim 44, wherein thecross-linkable polymer is selected from the group consisting ofpoly(ethyleneimine), poly(diallylammonium chloride), and poly(glycidylmethacrylate).
 60. A process according to claim 44, wherein thecross-linking agent is selected from the group consisting of ethyleneglycol diglycidyl ether, poly(propylene glycol) diglycidyl ether,1,3-dibromopropane, 1,4-dibromobutane, 1,5-dibromopentane,1,6-dibromohexane, α,α′-dibromo-p-xylene, α,α′-dichloro-p-xylene,1,4-dibromo-2-butene, piperazine, 1,4-diazabicyclo[2.2.2]octane,1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane,1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane,1,8-diaminooctane.
 61. A process according to claim 44, wherein aphotoinitiator is added prior to macroporous cross-linked gel formation.62. A process according to claim 61, wherein the photoinitiator isselected from the group consisting of2-hydroxy-1[4-(2-hydroxyethoxy)phenyl]2-hydroxy-2-methyl-1-propane-1-one,2,2-dimethoxy-2-phenylacetophenone (DMPA), benzophenone, benzoin andbenzoin ethers, dialkoxyacetophenones, hydroxyalkylphenones, andα-hydroxymethyl benzoin sulfonic esters.
 63. A process according toclaim 44, wherein a thermoinitiator is added prior to macroporouscross-linked gel formation.
 64. A process according to claim 63, whereinthe thermoinitiator is selected from the group consisting of1,1′-azobis(cyclohexanecarbonitrile), azobis(isobutyronitrile) (AIBN),potassium persulfate, ammonium persulfate, and benzoyl peroxide.
 65. Aprocess according to claim 44, wherein a porogen is added prior tomacroporous cross-linked gel formation.
 66. A process according to claim65 wherein the porogen is a poor solvent for the macroporouscross-linked gel.
 67. A process according to claim 44, wherein theformed macroporous cross-linked gel contains reactive functional groupsselected from the group consisting of epoxy, anhydride, azide, reactivehalogen, and acid chloride groups, to which functional groups are thenreacted.
 68. A process according to claim 44, wherein the formedmacroporous cross-linked gel contains groups that can be quaternizedthrough a subsequent reaction to give a charged macroporous cross-linkedgel.
 69. A process for chromatographic filtration of a solution orsuspension containing two or more species of different size that aredissolved or suspended in a fluid, which process comprises (a) passingthe fluid through a composite material as claimed in claim 31 so thatspecies of the smallest size pass through the composite material butlarger species do not pass through the composite material, and (b)changing an environmental condition to increase the size of the pores inthe macroporous gel so that species of a next larger size pass throughthe composite material.
 70. The process according to claim 69, whereinprocess step (b) is repeated to allow sequential elution of largerspecies.
 71. The process according to claim 69, wherein the species areselected from proteins, cells, aggregates and particles.
 72. The processaccording to claim 69, wherein the environmental condition is selectedfrom the group consisting of pH, ionic strength, temperature, lightintensity, and electrochemical current or a combination thereof.